Anti-p53 Antibodies

ABSTRACT

The present invention relates to nucleotide sequences which encode polypeptides of antibodies against the p53 protein in vertebrates, and to die polypeptides and antibodies (or fragments thereof) encoded by those nucleotide sequences. The invention also relates to nucleotide sequences and polypeptide sequences for use in the development of diagnostic and therapeutic compositions, and to methods of using those diagnostic and therapeutic compositions in the diagnosis and treatment of cancer, rheumatoid arthritis and other disease states which exhibit abnormalities of p53.

TECHNICAL FIELD

The present invention relates to nucleotide sequences which encodepolypeptides of antibodies against the p53 protein in vertebrates, andto the polypeptides and antibodies (or fragments thereof) encoded bythose nucleotide sequences. The invention also relates to nucleotidesequences and polypeptide sequences for use in the development ofdiagnostic and therapeutic compositions, and to methods of using thosediagnostic and therapeutic compositions in the diagnosis and treatmentof cancer, rheumatoid arthritis and other disease states which exhibitabnormalities of p53.

BACKGROUND OF THE INVENTION

The p53 gene is mutated in more than 50% of human tumours (1), Pointmutations in the central DNA binding domain are the most frequentlyobserved mutation (2), and result in loss of function due toconformational changes (3). The half life of the mutated protein isusually increased resulting in accumulation of p53 in tumour cells. Thisaccumulation of mutant protein is implicated as a factor in thedevelopment of an immune response to the protein in some cancer patients(4). The conformation of wild-type p53 is not static, with changesbetween the wild-type and mutant phenotype or conformation beinginduced, for instance, in vitro by buffer conditions, monoclonalantibodies, kinases and enzymes, or in vivo by kinases, phosphatases,and other p53 regulatory proteins.

Anti-P53 serum antibodies have been detected in up to 30% of individualswith cancer, and a range of different tumours Monoclonal antibodies(MAb) to p53 have been invaluable in investigating the function of p53and its role in tumorigenesis,

Molecular approaches to the generation of Mab offer several advantagesover traditional methods such as EBV transformation or hybridomatechnology. In part, this is because in humans, these traditionalmethods often result in a bias towards certain B cell populations andthe creation of cell lines which are unstable or producing only lowlevels of antibody (5). In contrast, molecular genetic approaches allowthe use of genetic material from any source of available B lymphocytesto create random combinations of cloned heavy and light chainimmunoglobulin genes.

Previous studies of the immune response against p53 in cancer patientshave relied on serum analysis. These studies have yielded importantinformation on the clinical significance, epitope dominance and the roleof protein overexpression in the development of the anti-p53 immuneresponse. However, several critical questions remain unanswered. To dateno human anti-p53 Mabs have been isolated either by conventional cellimmortalisation methods or molecular biological procedures Hence noinformation is available on human anti-p53 antibody V gene usage, thedegree of somatic mutation and structural features of the anti-p53antibodies Such information is critical to any meaningful understandingof the nature and significance of the humoral immune response to p53.

The present invention describes the isolation of anti-p53 antibodies.The nucleotide sequence and gene usage of these antibodies wereexamined. These antibodies are a rich resource for use in functionalstudies of the protein diagnostic assessment of p53 in normal anddisease states as well as in the development of to vaccines, includingidiotypic vaccines.

SUMMARY OF THE INVENTION

1. Nucleic Acid Encoding a Polypeptide of an Antibody or Fragmentthereof to p53.

According to a first embodiment of the invention, there is provided anisolated and purified nucleic acid sequence comprising a polynucleotidesequence encoding a polypeptide of an antibody (or fragment thereof),wherein said antibody (or fragment thereof) has binding affinity to ap53 protein or a portion thereof in vertebrates, and wherein saidnucleic acid sequence is obtained from a vertebrate host expressing animmune response against a naturally-occurring disease.

Typically, the immune response is characterised by expression of atleast one p53 antibody.

Typically, the nucleic acid molecule comprises a polynucleotide sequenceencoding an Fab antibody fragment (or fragment thereof) having bindingaffinity to a p53 protein or a portion thereof in vertebrates.

According to a second embodiment of the invention, there is provided anisolated and purified nucleic acid molecule comprising a polynucleotidesequence selected from the group consisting of SEQ ID Nos 1-30.

The following features relate to the first and second embodiments of theinvention.

Typically, the nucleic acid molecule corresponds to a DNA or RNAmolecule Generally, the nucleic acid molecule comprises a polynucleotidesequence(s), or an analogue thereof, encoding an antibody fragment orother immunologically active fragments thereof, such as complementaritydetermining regions, wherein the antibody (or fragment thereof) hasbinding affinity to a p53 protein or a portion thereof in vertebrates.

Typically, the antibody fragment has functional antigen-binding domainsEven more typically, the antibody fragment may exist in a form selectedfrom the group consisting of Fv, F_(ab), F(ab)₂, scFv (single chain Fv),dAb (single domain antibody), bi-specific antibodies, diabodies andtriabodies.

Typically, the antibody (or fragment thereof) has binding affinity to ap53 protein or a portion thereof. More typically, the antibody (orfragment thereof) has binding affinity for residues of one or more ofthe N-terminus, the C-terminus or the central domain of a p53 protein ora portion thereof Even more typically, the antibody (or fragmentthereof) has binding affinity for residues of the N-terminus of a p53protein or a portion thereof. Yet more typically, the antibody (orfragment thereof) has binding affinity for residues about 10 to about55. Still more typically, the antibody (or fragment thereof) has bindingaffinity for residues about 10 to about 25, or about 40 to about 50, orabout 27 to about 44 of the N-terminus of a p53 protein or portionthereof or about 40 to about 44 of the N-terminus of a p53 protein orportion thereof. Even more typically, the antibody (or fragment thereof)has binding affinity for residues about 27 to about 44 of the N-terminusof a p53 protein or a portion thereof. Still more typically, theantibody (or fragment thereof) has binding affinity for residues about40 to about 44 of the N-terminus of a p53 protein or a portion thereof.

Even more typically, the antibody (or fragment thereof) has bindingaffinity to residues of the central domain of a p53 protein or a portionthereof.

Typically, the nucleic acid molecule comprises a polynucleotide sequenceencoding a polypeptide of an antibody (or fragment thereof) havingbinding affinity to a p53 protein or a portion thereof in vertebrates,wherein said polynucleotide sequence encodes an immunoglobulin lightchain variable region polypeptide or an immunoglobulin heavy chainvariable region polypeptide.

More typically, the nucleic acid molecule comprises a polynucleotidesequence encoding a polypeptide of an antibody (or fragment thereof)having binding affinity to a p53 protein or a portion thereof invertebrates, wherein said nucleic acid molecule comprises a first,polynucleotide sequence encoding an immunoglobulin light chain variableregion polypeptide, and a second polynucleotide sequence encoding animmunoglobulin heavy chain variable region polypeptide.

Typically, the polynucleotide sequence encoding the immunoglobulin lightchain variable region comprises polynucleotide sequencers) encodingimmunoglobulin light chain variable (V region) and joining (J region)segments.

Typically, the polynucleotide sequence encoding the immunoglobulin heavychain variable region polypeptide comprises polynucleotide sequence(s)encoding immunoglobulin heavy chain variable (V region), diversity (Dregion) and joining (J region) segments.

More typically, the nucleic acid molecule also comprises apolynucleotide sequencers) encoding one or more immunoglobulin constantregions operably linked with the immunoglobulin heavy chain variable orimmunoglobulin light chain region(s). Even more typically, at least oneof the immunoglobulin constant regions may be derived from a differentsource than the source from which the immunoglobulin variable region wasderived. Still more typically, the source from which the immunoglobulinconstant region is derived is human.

Typically, the p53 protein or a portion thereof is encoded by a wildtype or mutant p53 gene.

Typically, the vertebrate is selected from the group consisting ofhuman, non-human primate, murine, bovine, ovine, equine, caprine,leporine, avian, feline and canine. More typically, the vertebrate ishuman, non-human primate or murine. Even more typically, the vertebrateis human.

Typically, the nucleic acid molecule also includes within its scope ananalogue of the polynucleotide sequence defined in accordance with thefirst or second embodiments of the invention, wherein said analogueencodes a polypeptide having a biological activity which is functionallythe same as the polypeptide(s) encoded by the polynucleotide sequencedefined in accordance with the first or second embodiments of theinvention, wherein said polynucleotide sequence can be located andisolated using standard techniques in molecular biology, without unduetrial and experimentation.

Typically, the nucleic acid molecule also includes within its scope ananalogue of the polynucleotide sequence defined in accordance with thefirst or second embodiments of the invention, which has at least 45%homology to the polynucleotide sequences so defined. More typically, theanalogue of the polynucleotide sequences has at least 55% homology,still more typically the analogue has at least 60% homology, even moretypically, the analogue has at least 75% homology, still more typically,the analogue has at least 85% homology, and yet still more typically,the analogue has at least 90% homology, and yet even still moretypically, the analogue has at least 95-99% homology to thepolynucleotide sequences so defined.

The degree of homology between two nucleic acid sequences may bedetermined by means of computer programs known in the art such as GAPprovided in the GCG program package (Program Manual for the WisconsinPackage, Version 8, August 1996, Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D.,(1970), Journal of Molecular Biology, 48; 443-453), Using GAP with thefollowing settings for DNA sequence comparison: GAP creation penalty of5.0 and GAP extension penalty of 0.3.

Antibody sequences may be aligned to each other using the Pileupalignment software, available as part of the GCG program package, using,for instance, the default settings of gap creation penalty of 5 and gapwidth penalty of 0.3.

Typically, the nucleic acid molecule also includes within its scope ananalogue of the polynucleotide sequence defined in accordance with thefirst or second embodiments of the invention, wherein said analogue iscapable of hybridising to the polynucleotide sequences under conditionsof low stringency. More typically, low stringency hybridisationconditions correspond to hybridisation performed at 50° C. in 6×SSC.

Suitable experimental conditions for determining whether a given nucleicacid molecule hybridises to a specified nucleic acid may involvepresoaking of a filter containing a relevant sample of the nucleic acidto be examined in 5×SSC for 10 min, and prehybridisation of the filterin a solution of 5×SSC, 5×Denhardt's solutions 0.5% SDS and 100 μg/ml ofdenatured sonicated salmon sperm DNA, followed by hybridisation in thesame solution containing a concentration of 10 ng/ml of a³²P-dCTP-labeled probe for 12 hours at approximately 45° C., inaccordance with the hybridisation methods as described in Sambrook etal. (1989; Molecular Cloning, A Laboratory Manual, 2nd edition, ColdSpring Harbour, N.Y.).

The filter is then washed twice for 30 minutes in 2×SSC, 0 5% SDS atleast 55° C. (low stringency), at least 60° C. (medium stringency), atleast 65° C. (medium/high stringency), at least 70° C. (highstringency), or at least 75° C. (very high stringency). Hybridisationmay be detected by exposure of the filter to an x-ray film.

Further, there are numerous conditions and factors, well known to thoseskilled in the art, which may be employed to alter the stringency ofhybridisation. For instance, the length and nature (DNA. RNA, basecomposition) of the nucleic acid to be hybridised to a specified nucleicacid; concentration of salts and other components, such as the presenceor absence of formamide dextran sulfate, polyethylene glycol etc; andaltering the temperature of the hybridisation and/or washing steps.

Further, it is also possible to theoretically predict whether or not twogiven nucleic acid sequences will hybridise under certain specifiedconditions Accordingly, as an alternative to the empirical methoddescribed above, the determination as to whether an analogous nucleicacid sequence will hybridise to the nucleic acid molecule in accordancewith the first or second embodiments of the invention, can be based on atheoretical calculation of the T_(m) (melting temperature) at which twoheterologous nucleic acid sequences with known sequences will hybridiseunder specified conditions such as salt concentration and temperature.

In determining the melting temperature for heterologous nucleic acidsequences (T_(m(hetero))) it is necessary first to determine the meltingtemperature (T_((homo))) for homologous nucleic acid sequence. Themelting temperature (T_(m(homo))) between two fully complementarynucleic acid strands (homoduplex formation) may be determined inaccordance with the following formula, as outlined in Current Protocolsin Molecular Biology, John Wiley and Sons, 1995, as:

T_(m(homo))=81.5° C.+16.6(log M)+0.41(% GC)−0 61 (% form)−500/L

M=denotes the molarity of monovalent cations,

% GC=% guanine (G) and cytosine (C) of total number of bases in thesequence,

% form=% formamide in the hybridisation buffer, and

L=the length of the nucleic acid sequence.

T_(m) determined by the above formula is the T_(m) of a homoduplexformation (T_(m(homo))) between two fully complementary nucleic acidsequences. In order to adapt the T_(m) value to that of two heterologousnucleic acid sequences, it is assumed that a 1% difference in nucleotidesequence between two heterologous sequences equals a 1° C. decrease inT_(m). Therefore, the T_(m(hetero)) for the heteroduplex formation isobtained through subtracting the homology % difference between theanalogous sequence in question and the nucleotide probe described abovefrom the T_(m(homo)).

Typically, the nucleic acid molecule also includes within its scope ananalogue of the polynucleotide sequence defined in accordance with thefirst or second embodiments of the invention which because of thedegeneracy of the genetic code, does not hybridise with thepolynucleotide sequence defined in accordance with the second embodimentof the invention, but which encodes a polypeptide of an antibody (orfragment thereof) having binding affinity to a p53 protein or a portionthereof in vertebrates.

Typically the nucleic acid molecule as defined in accordance with thefirst or second embodiments of the invention also includes within itsscope a nucleic acid molecule which is an oligonucleotide fragment ofthe polynucleotide sequence defined in accordance with the first orsecond embodiments of the invention.

Typically, the oligonucleotide fragment is between about 10 to about 100nucleotides in length. More typically, the oligonucleotide fragment isbetween about 10 to about 75 nucleotides in length Even more typically,the oligonucleotide fragment is between about 15 to about 50 nucleotidesin length. Even more typically still, the oligonucleotide fragment isbetween about 15 to about 30 nucleotides in length. Yet still moretypically, the oligonucleotide fragment is between about 5 to about 25nucleotides in length

2. Polypeptide of an Antibody or Fragment thereof to p53 and/or Antibodyor Fragment thereof to p53.

According to a third embodiment of the invention, there is provided apolypeptide of an antibody (or fragment thereof) having binding affinityto a p53 protein or a portion thereof in vertebrates, wherein saidpolypeptide is obtained from a vertebrate host expressing an immuneresponse against a naturally-occurring disease.

Typically, the immune response is characterised by expression of atleast one p53 antibody.

According to a fourth embodiment of the invention, there is provided apolypeptide wherein said polypeptide is encoded by the nucleic acidmolecule defined in accordance with the first or second embodiments ofthe invention.

According to a fifth embodiment of the invention, there is provided apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID Nos 31-60.

The following features relate to the third, fourth and fifth embodimentsof the invention.

Typically, the polypeptide comprises functional antigen-binding domains,that is, heavy and light chain variable domains. Even more typically,the polypeptide of the antibody (or fragment thereof) may exist in aform selected from the group consisting of: Fv, F_(ab), F(ab)₂, scFv(single chain Fv), dAb (single domain antibody), bi-specific antibodies,diabodies and triabodies.

Typically, the polypeptide has binding affinity to a p53 protein or aportion thereof. More typically, the antibody has binding affinity forresidues of one or more of the N-terminus, the C-terminus or the centraldomain of a p53 protein or a portion thereof Even more typically, theantibody has binding affinity for residues of the N-terminus of a p53protein or a portion thereof Yet more typically, the antibody hasbinding affinity for residues about 10 to about 55. Still moretypically, the antibody has binding affinity for residues about 10 toabout 25, or about 40 to about 50, or about 27 to about 44 of theN-terminus of a p53 protein or portion thereof or about 40 to about 44of the N-terminus of a p53 protein or portion thereof. Even moretypically, the antibody has binding affinity for residues about 27 toabout 44 of the N-terminus of a p53 protein or a portion thereof. Stillmore typically, the antibody has binding affinity for residues about 40to about 44 of the N-terminus of a p53 protein or a portion thereof.

Even more typically, the antibody has binding affinity to residues ofthe central domain of a p53 protein or a portion thereof.

Typically, the polypeptide corresponds to an immunoglobulin light chainvariable region polypeptide or an immunoglobulin heavy chain variableregion polypeptide.

More typically, the polypeptide comprises a first polypeptide whichcorresponds to an immunoglobulin light chain variable regionpolypeptide, and a second polypeptide which corresponds to animmunoglobulin heavy chain variable region polypeptide.

Typically, the immunoglobulin light chain variable region polypeptidecomprises immunoglobulin light chain variable (V region) and joining (Jregion) segments.

Typically, the immunoglobulin heavy chain variable region polypeptidecomprises immunoglobulin heavy chain variable (V region), diversity (Dregion) and joining (J region) segments.

More typically, the polypeptide also comprises polypeptide(s) whichcorrespond to an immunoglobulin constant region(s) operably linked withthe immunoglobulin light or heavy chain variable region(s). Even moretypically, at least one of the constant regions may be derived from adifferent source than the source from which the variable region wasderived. Still more typically, the source from which the constant regionis derived is human.

Typically, the polypeptide includes within its scope a peptide fragmentof the polypeptide of the third, fourth or fifth embodiment, whereinsaid peptide fragment may or may not have binding affinity to a p53protein or a portion thereof.

Typically, the peptide fragment of the polypeptide is between about 5 toabout 50 contiguous amino acids. More typically, between about 5 toabout 35 contiguous amino acids. Even more typically, between about 5 toabout 30 contiguous amino acids. Still more typically, between about 5to about 25 contiguous amino acids. Yet still more typically, betweenabout 8 to about 20 contiguous amino acids.

Typically, the polypeptide also includes within its scope a homologouspolypeptide of the polypeptide defined in accordance with the third,fourth and fifth embodiments of the invention, which has at least 35%homology to the polypeptide sequences so defined. More typically, thehomologue of the polypeptide sequences has at least 45% homology, stillmore typically the homologue has at least 65% homology, even moretypically, the homologue has at least 75% homology, still moretypically, the homologue has at least 85% homology, and yet still moretypically, the homologue has at least 90% homology, and yet even stillmore typically, the homologLie has at least 95-99% homology to thepolypeptide sequences so defined.

As applied to polypeptides, the degree of homology between twopolypeptide sequences when optimally aligned, may be determined throughthe use of computer alignment programs known in the art such as, forexample, BLAZE (Intelligenetics) GAP, BESTFIT, ALIGN, using default gapweights. One specific example is the GAP program as provided in the GCGprogram package (Program Manual for the Wisconsin Package, Version 8,August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wis.,USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal ofMolecular Biology, 48, 443-453), using the following settings forsequence comparison; GAP creation penalty of 5.0 and GAP extensionpenalty of 0.3.

According to a sixth embodiment of the invention, there is provided anantibody (or fragment thereof), wherein said antibody (or fragmentthereof) has binding affinity to a p53 protein or a portion thereof invertebrates and wherein said antibody is obtained from a vertebrate hostexpressing an immune response against a naturally-occurring disease.

Typically, the immune response is characterised by expression of atleast one p53 antibody

Typically, the antibody (or fragment thereof) having binding affinity toa p53 protein or a portion thereof in vertebrates, corresponds to the anantibody (or fragment thereof) encoded by the nucleic acid molecule asdefined in accordance with the first or second embodiments of theinvention.

According to a seventh embodiment of the invention, there is provided anantibody (or fragment thereof) having binding affinity to a p53 proteinor a portion thereof in vertebrates, wherein said antibody (or fragmentthereof) is comprised of the polypeptide as defined in accordance withthe third, fourth or fifth embodiment of the invention.

Typically, the antibody (or fragment thereof) having binding affinity toa p53 protein or a portion thereof in vertebrates, corresponds to thepolypeptide as defined in accordance with the third, fourth or fifthembodiment of the invention.

Typically, as described throughout the specification, the antibody maybe a whole antibody, or an antibody fragment, or other immunologicallyactive fragments thereof, such as complementarity determining regions.More typically, the antibody fragment has functional antigen-bindingdomains, that is, heavy and light chain variable domains. Even moretypically, the antibody fragment may exist in a form selected from thegroup consisting of: Fv, F_(ab), F(ab)₂, scFv (single chain Fv), dAb(single domain antibody), bi-specific antibodies, diabodies andtriabodies.

The following features relate to the sixth and seventh embodiments ofthe invention.

Typically, the antibody (or fragment thereof) is a polyclonal ormonoclonal antibody More typically, the antibody (or fragment thereof)is a monoclonal antibody. Even more typically, the monoclonal antibodyis generated using molecular genetic, hybridoma or EBV (Epstein-Barrvirus) transformation technology. Even more typically, the monoclonalantibody may be generated using recombinant antibody techniques throughscreening a combinatorial antibody library or phage display technology.

Typically, the antibody (or fragment thereof) has binding affinity to ap53 protein or a portion thereof. More typically, the antibody (orfragment thereof) has binding affinity for residues of one or more ofthe N-terminus, the C-terminus or the central domain of a p53 protein ora portion thereof. Even more typically, the antibody (or fragmentthereof) has binding affinity for residues of the N-terminus of a p53protein or a portion thereof. Yet more typically, the antibody (orfragment thereof) has binding affinity for residues about 10 to about55. Still more typically, the antibody (or fragment thereof) has bindingaffinity for residues about 10 to about 25, or about 40 to about 50, orabout 27 to about 44 of the N-terminus of a p53 protein or portionthereof or about 40 to about 44 of the N-terminus of a p53 protein orportion thereof. Even more typically, the antibody (or fragment thereof)has binding affinity for residues about 27 to about 44 of the N-terminusof a p53 protein or a portion thereof. Still more typically, theantibody (or fragment thereof) has binding affinity for residues about40 to about 44 of the N-terminus of a p53 protein or a portion thereof.

Even more typically, the antibody has binding affinity to residues ofthe central domain of a p53 protein or a portion thereof.

The following features relate to the first through to seventhembodiments of the invention.

Typically, the disease is selected from the group consisting of: cancer,rheumatoid arthritis and coronary heart disease. More typically, thedisease is cancer. Typically, the cancer is selected from the groupconsisting of carcinogenic tumours, tumours of epithelial origin, suchas colo-rectal cancer, breast cancer, lung cancer, head and necktumours, hepatic cancer, pancreatic cancer, ovarian cancer, gastriccancer, brain cancer, bladder cancer, prostate cancer andurinary/genital tract cancer, oesophageal cancer; mesenchymal tumours,such as sarcoma: and haemopoietic tumours, such as B cell lymphoma.

According to an eighth embodiment of the invention, there is provided avector comprising the nucleic acid molecule as defined in accordancewith the first or second embodiments of the invention.

Typically, the vector is a shuttle or expression vector. More typically,the vector is selected from the group consisting of, viral, plasmid,bacteriophage, phagemid cosmid, bacterial artificial chromosome, andyeast artificial chromosome.

Typically, the vector is a plasmid and may be selected from the groupconsisting of pBR322, M13mp18, pUC 18 and pUC 19.

Typically, the vector is a bacteriophage and may be selected from λgt10and λgt11 or phage display vectors. More typically, the phage displayvector is selected from vectors derived from pCOMB vectors. Even moretypically, the phage display vector is of the MCO group, which forexample, may include MCO1, MCO3 and MGO6 vectors. Still more typically,the vector is MCO3.

Typically, the vector is a mammalian expression vector, such aspG1D102-MCO or pKN100-MCO.

Typically, the vector includes expression control sequences, such as anorigin of replication, a promoter an enhancer, and necessary processinginformation sites, such as ribosome binding sites. RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.

More typically, the MCO vector contains, amongst others, sequencesselected from the group consisting of: polypeptide tag, amber codon,genelll, heavy and light chain specific multicloning site, ompA and/orpelB leader sequences, subtilisin cleavage site, and/or 6 histidine tag.

Still more typically, the vector may include selection markers to permitdetection of those cells transformed with the desired polynucleotidesequences.

Typically, the vector may include heterologous coding sequence orsequences to permit the expression of a fusion protein comprising thepolypeptide of the third, fourth or fifth embodiments.

According to a ninth embodiment of the invention there is provided ahost cell transformed with the vector as defined in accordance with theeighth embodiment of the invention.

Typically, the host cells are procaryotic or eucaryotic in nature.

More typically the procaryotic host cells include bacteria, and examplesof such bacteria include: E. coli, Bacillus, Streptomyces, Pseudomonas,Salmonella, and Serratia.

More typically, the eucaryotic host cells may be selected from the groupconsisting of: yeast, fungi, plant insect cells and mammalian cells,either in vivo or in tissue culture. Examples of mammalian cellsinclude: CHO cell lines, COS cell lines, HeLa cells, L cells, murine 3T3cells, c6 glioma cells and myeloma cell lines.

Still more typically, the eucaryotic host cells are CHO DG44 cells.

According to a tenth embodiment of the invention, there is provided avertebrate comprising a host cell as defined in accordance with theninth embodiment of the invention, wherein said vertebrate does notinclude humans.

3. Pharmaceutical/Therapeutic and Diagnostic Compositions

According to an eleventh embodiment of the invention, there is provideda pharmaceutical composition comprising the polypeptide, or peptidefragment, as defined in accordance with the third, fourth or fifthembodiments of the invention, or an antibody (or fragment thereof) asdefined in accordance with the sixth or seventh embodiments of theinvention, together with a pharmaceutically acceptable carrier, adjuvantand/or diluent.

Typically, the antibody present in the pharmaceutical composition mayexist as a whole antibody, or be present as an antibody fragment orother immunologically active fragments thereof such as complementaritydetermining regions. More typically, the antibody fragment hasfunctional antigen-binding domains, that is, heavy and light chainvariable domains. Even more typically, the antibody fragment may existin a form selected from the group consisting of: Fv, F_(ab), F(ab)₂,scFv (single chain Fv), dAb (single domain antibody), bi-specificantibodies, diabodies and triabodies.

Typically, the polypeptide, peptide fragment, or antibody or fragmentthereof present in the pharmaceutical composition may also exist in aform selected from the group consisting of: polypeptide/chelate,polypeptide/drug, polypeptide/prodrug, polypeptide/toxin,polypeptide/imaging marker, antibody/chelate, antibody/drug,antibody/prodrug, antibody/toxin and antibody/imaging marker.

More typically, the chelate may be selected from the group consistingof: ⁹⁰Y, ¹³¹I and ¹⁸⁸Re.

More typically, the drug may be a cytotoxic drug. Even more typically,the cytotoxic drug may be selected from the group consisting of:adriamycin, melphalan, cisplatin, taxol, fluorouricil, cyclophosphamideand others known to those of skill in the art such as those included in“The Chemotherapy Source Book”, M. C. Perry Williams and Wilkins, 2^(nd)Ed, 1996), the entire contents of which are incorporated herein byreference.

More typically, the prodrug may be antibody directed prodrug therapy orADEPT.

More typically, the toxin may be selected from the group consisting of:ricin, abrin, Diptheria toxin and Pseudomonas endotoxin (PE 40).

Typically, the imaging marker includes substances which can be detectedby a gamma scanner or hand held gamma probe, and substances which can bedetected by nuclear magnetic resonance imaging using a nuclear magneticresonance spectrometer.

More typically, the imaging marker which may be detected using a gammascanner include imaging markers selected from the group consisting of¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷ Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, and ^(99m)Tc.

Typically, the imaging marker which can be detected using a nuclearmagnetic resonance spectrometer is gadolinium.

Typically, the pharmaceutical composition in accordance with theeleventh embodiment of the invention may also include cytokines, suchas, G-CSF, GM-CSF, interleukins.

Typically, the pharmaceutical composition in accordance with theeleventh embodiment of the invention may also include an adjuvant suchas mannan.

According to a twelfth embodiment of the invention, there is provided avaccine, wherein said vaccine comprises a nucleic acid molecule asdefined in accordance with the first or second embodiments of theinvention, or a fragment thereof, or a polypeptide, or peptide fragment,as defined in accordance with the third, fourth or fifth embodiments ofthe invention, or an antibody or fragment thereof as defined inaccordance with the sixth or seventh embodiments of the inventiontogether with a pharmaceutically acceptable carrier adjuvant and/ordiluent.

Typically, the vaccine is an idiotypic vaccine.

Typically, the antibody (or a fragment thereof) of the present inventionmay be used as an idiotypic immunogen When used in this manner, theantibody (or a fragment thereof) of the present invention may functionas an immunogen and elicit a second antibody (Ab2) and T cell (T₂)response against idiotopes of the original antibody (Ab1) Ab2 antibodiescan bind to epitopes on the original antibody including the antigenbinding site (idiotype). The anti-idiotypic antibody, Ab2, canspontaneously induce anti-anti-idiotypic antibodies (Ab3) as well as Tcells (T₃) which may recognise the same epitope as Ab1. Since the firstantibody binds both the p53 epitope and Ab2, Ab2 mimics the structure ofthe antigenic epitope (on p53). A proportion of Ab3 antibodies bind tothe same epitope as the original antibody (Ab1), and may augment andprolong the efficacy of the original antibody. Induction of thisanti-idiotypic network results in protection from metastases partlythrough the induction of p53-specific CTLs.

Alternatively, a vaccine composition containing a peptide fragment ofthe polypeptide of the present invention may be prepared by synthesis ofa peptide. For example, the peptide may comprise selected amino acidregions of the COR and/or FR of the polypeptide of the invention.Typically, the peptide fragment of the polypeptide of the presentinvention may or may not have binding affinity for a p53 protein or aportion thereof in vertebrates.

Typically, the vaccine is formulated for administration via an oral,inhalation, topical or parenteral route. More typically the route ofadministration is parenteral.

According to a thirteenth embodiment of the invention, there is provideda method for inducing an immune response against disease in avertebrate, comprising administering to said vertebrate animmunologically effective amount of the polypeptide (or peptide fragmentthereof) as defined in accordance with the third fourth or fifthembodiments of the invention, or an antibody (or fragment thereof) asdefined in accordance with the sixth or seventh embodiments of theinvention or a pharmaceutical composition as defined in accordance withthe eleventh embodiment of the invention or a vaccine as defined inaccordance with the twelfth embodiment of the invention.

Typically, the polypeptide, or peptide fragment or antibody (or fragmentthereof) as administered in accordance with the thirteenth embodiment ofthe invention, is administered together with a pharmaceuticallyacceptable carrier, adjuvant and/or diluent.

Typically, the polypeptide, or peptide fragment or antibody (or fragmentthereof) as administered in accordance with the thirteenth embodiment ofthe invention, may also be simultaneously or sequentially administeredwith cytokines, such as: G-CSF, GM-CSF, interleukins.

Typically, the pharmaceutical composition in accordance with thethirteenth embodiment of the invention may also include an adjuvant,such as mannan.

According to a fourteenth embodiment of the invention, there is providedthe polypeptide (or peptide fragment thereof) as defined in accordancewith the third, fourth or fifth embodiments of the invention or anantibody (or fragment thereof) as defined in accordance with the sixthor seventh embodiments of the invention, or a pharmaceutical compositionas defined in accordance with the eleventh embodiment of the invention,or a vaccine as defined in accordance with the twelfth embodiment of theinvention, when used in inducing an immune response against disease in avertebrate.

According to a fifteenth embodiment of the invention, there is providedthe use of the polypeptide (or peptide fragment thereof) as defined inaccordance with the third, fourth or fifth embodiments of the invention,or an antibody (or fragment thereof) as defined in accordance with thesixth or seventh embodiments of the invention in the preparation of avaccine for inducing an immune response against disease in a vertebrate.

According to a sixteenth embodiment of the invention, there is provideda method for inducing an immune response against disease in avertebrate, comprising administering to said vertebrate animmunologically effective amount of the vaccine as defined in accordancewith the twelfth embodiment of the invention.

According to a seventeenth embodiment of the invention, there isprovided a vaccine as defined in accordance with the twelfth embodimentof the invention when used in inducing an immune response againstdisease in a vertebrate.

According to an eighteenth embodiment of the invention, there isprovided a method for the treatment and/or prophylaxis of disease in avertebrate in need of said treatment and/or prophylaxis, wherein saidmethod comprises administering a therapeutically effective amount of thepolypeptide (or peptide fragment thereof) in accordance with the third,fourth or fifth embodiments of the invention, or an antibody (orfragment thereof) in accordance with the sixth or seventh embodiments ofthe invention, or a pharmaceutical composition as defined in accordancewith the eleventh embodiment of the invention, or a vaccine as definedin accordance with the twelfth embodiment of the invention.

According to a nineteenth embodiment of the invention, there is providedthe polypeptide (or peptide fragment thereof) in accordance with thethird, fourth or fifth embodiments of the invention, or an antibody (orfragment thereof) in accordance with the sixth or seventh embodiments ofthe invention, or a pharmaceutical composition as defined in accordancewith the eleventh embodiment of the invention or a vaccine as defined inaccordance with the twelfth embodiment of the invention when used in thetreatment and/or prophylaxis of disease in a vertebrate in need of saidtreatment and/or prophylaxis.

According to a twentieth embodiment of the invention, there is provideduse of the polypeptide (or peptide fragment thereof) in accordance withthe third, fourth or fifth embodiments of the invention or an antibody(or fragment thereof) in accordance with the sixth or seventhembodiments of the invention, or a pharmaceutical composition as definedin accordance with the eleventh embodiment of the invention, or avaccine as defined in accordance with the twelfth embodiment of theinvention in the preparation of a medicament for the treatment and/orprophylaxis of disease in a vertebrate in need of said treatment and/orprophylaxis.

Typically, the disease is selected from the group consisting of: cancer,rheumatoid arthritis and coronary heart disease. More typically, thedisease is cancer.

Typically, the cancer is selected from the group consisting of:carcinogenic tumours; tumours of epithelial origin, such as coo-rectalcancer, breast cancer, lung cancer, head and neck tumours, hepaticcancer, pancreatic cancer, ovarian cancer, gastric cancer, brain cancer,bladder cancer, prostate cancer and urinary/genital tract cancer,oesophageal cancer: mesenchymal tumours, such as sarcoma: andhaemopoietic tumours, such as B cell lymphoma.

4. An Antibody/Nucleic Acid Based Method and Kit for Detecting p53

According to a twenty-first embodiment of the invention, there isprovided a diagnostic kit for the detection of polypeptides encoded bythe p53 gene in vertebrates, comprising the antibody (or fragmentthereof) as defined in accordance with the sixth or seventh embodimentsof the invention, together with a diagnostically acceptable carrierand/or diluent.

Typically, the kit may comprise the following containers;

(a) a first container containing the antibody (or fragment thereof) asdefined in accordance with the sixth or seventh embodiments of theinvention, and;

(b) a second container containing a conjugate comprising a bindingpartner of the antibody (or fragment thereof), together with adetectable label.

More typically, the kit may further comprise one or more othercontainers, containing other components, such as wash reagents, andother reagents capable of detecting the presence of bound antibodiesEven more typically, the detection reagents may include: labelled(secondary) antibodies, or where the antibody (or fragment thereof) ofthe present invention is itself labelled, the compartments may compriseantibody binding reagents capable of reacting with the labelled antibody(or fragment thereof) of the present invention.

According to a twenty-second embodiment of the invention, there isprovided a method for screening for a disease in a vertebrate comprising

(a) contacting a sample from a vertebrate with a nucleic acid probe, and

(b) detecting hybridisation between the nucleic acid sample and thepolynucleotide sequence.

Typically, hybridisation as compared to non-hybridisation is indicativeof disease. Typically, the disease is cancer.

Typically, the nucleic acid probe corresponds to a portion of thepolynucleotide sequence as defined in accordance with the first orsecond embodiments of the invention which is capable of selectivelyhybridising to nucleic acid from a sample.

Typically, hybridisation may occur and be detected through techniquesthat are routine and standard amongst those skilled in the art, andinclude southern and northern hybridisation, polymerase chain reaction(PCR) and ligase chain reaction (LCR) amplification.

Various low, medium or high stringency hybridisation levels may be used,depending on the specificity and selectivity desired.

According to a twenty-third embodiment of the invention, there isprovided a method for screening for a disease in a vertebratecomprising:

(a) contacting a sample from a vertebrate with the antibody (or fragmentthereof) defined in accordance with the sixth or seventh embodiments ofthe invention, and

(b) detecting the presence of the antibody (or fragment thereof) boundto a p53 polypeptide.

Typically, altered levels of the p53 polypeptide in the sample ascompared to normal levels indicate disease. Typically, the disease iscancer.

5. Gene Therapy

According to a twenty-fourth embodiment of the invention, there isprovided a method of gene therapy, wherein said method comprises:

(a) inserting a nucleic acid molecule as defined in accordance with thefirst or second embodiments of the invention or a vector as defined inaccordance with the eighth embodiment of the invention into a host cell

(b) expressing the nucleic acid molecule in the transformed cell.

Typically, the nucleic acid molecule or vector is inserted using methodsselected from the group consisting of microinjection, CaPO₄precipitation electroporation lipofection/iliposome fusion particlebombardment and coupling the nucleic acid to chemically modifiedproteins.

Typically the nucleic acid molecule or vector is inserted into thenucleus of a host cell.

Typically an expression vector containing the nucleic acid molecule isinserted into cells the cells are grown in vitro and then infused inlarge numbers into patients. More typically, expression vectors derivedfrom viruses such as retroviruses, vaccinia virus, adenovirus,adeno-associated virus, herpes viruses, several RNA viruses, or bovinepapilloma virus, may be used for delivery of the nucleic acid into thetargeted cell population. More typically, the targeted cell populationcomprises tumour cells.

6. Preparing Antibody (or Fragment thereof) having Binding Affinity to ap53 Protein or a Portion thereof in Vertebrates

According to a twenty-fifth embodiment of the invention, there isprovided a process for preparing an antibody (or fragment thereof)having binding affinity to a p53 protein or a portion thereof invertebrates, wherein said process comprises:

(a) isolating from a vertebrate a nucleic acid molecule as defined inaccordance with the first or second embodiments of the invention;

(b) cloning said nucleic acid molecule into a vector;

(c) constructing an antibody fragment library, and

(d) screening said library for clones expressing the antibody ofinterest.

Typically, the antibody (or fragment thereof) as prepared by the processas defined in accordance with the twenty-fifth embodiment of theinvention has binding affinity to a p53 protein or a portion thereof invertebrates.

Typically, the nucleic acid sample is obtained from individualssuffering a disease associated with the expression of p53, who expressantibodies reactive with p53. More typically, the disease is selectedfrom the group consisting of: cancer, rheumatoid arthritis and coronaryheart disease. Even more typically, the disease is cancer. Still moretypically, the cancer is selected from the group consisting of:carcinogenic tumours; tumours of epithelial origin, such as colo-rectalcancer breast cancer, lung cancer, head and neck tumours hepatic cancer,pancreatic cancer, ovarian cancer, gastric cancer, brain cancer, bladdercancer, prostate cancer and urinary/genital tract cancer, oesophagealcancer; mesenchymal tumours, such as sarcoma and haemopoietic tumours,such as B cell lymphoma.

Typically, the nucleic acid sample is taken from an organ sufferingfrom, or a collection point for expression of, the disease. Even moretypically, the organ is a lymph node.

Typically, the nucleic acid sample is comprised of polynucleotidesequences in accordance with the first or second embodiments of theinvention.

Typically, the nucleic acid sample is mRNA. More typically, the clone isprepared through RT-PCR (reverse transcriptase PCR) and cloned into asuitable vector.

Typically, the vector is a phage display vector. More typically, thevector is selected from the group consisting of: MCO1, MCO3 and MCO6.Even more typically, the vector is MCO1.

Typically, nucleic acid clones are packaged into the phage displaylibrary to produce a primary antibody library. More typically, the phagedisplay library was amplified by panning against recombinant p53, andselected recombinant antibodies obtained.

Typically, the antibody library represents clones expressing an antibodyfragment, wherein said antibody has binding affinity to a p53 protein ora portion thereof. More typically, the antibody fragment is an F_(ab)fragment. Even more typically, the recombinant antibody fragment ispurified. Still more typically, the antibody fragment has bindingaffinity to a p53 protein or a portion thereof.

Typically, the antibody (or fragment thereof) having binding affinity toa p53 protein or a portion thereof is as defined in accordance with thesixth or seventh embodiments of the invention.

According to a twenty-sixth embodiment of the invention, there isprovided a method of locating a nucleotide sequence encoding apolypeptide of an antibody (or fragment thereof) having binding affinityto a p53 protein or portion thereof in vertebrates, using the nucleicacid molecule of the first or second embodiments of the invention.

Typically, the method comprises,

-   -   (a) contacting a biological sample with a nucleic acid molecule        of the first or second embodiments of the invention; and    -   (b) identifying nucleotide sequences in the biological sample        which hybridise to said nucleic acid molecule

Typically, step (a) is performed under conditions which promotehybridisation of homologous sequences, which conditions are well knownto those of skill in the art.

Specifically contemplated in this embodiment of the invention is amethod of locating a nucleotide sequence encoding a polypeptide of anantibody (or fragment thereof) wherein said antibody has bindingaffinity to a p53 protein or portion thereof in vertebrates.

Typically, the method is a method of locating a nucleotide sequenceencoding a polypeptide of an antibody (or fragment thereof) havingbinding affinity to residues of one or more of the N-terminus theC-terminus or the central domain of a p53 protein or a portion thereof.

More typically, the method is a method of locating a nucleotide sequenceencoding a polypeptide of an antibody (or fragment thereof) havingbinding affinity to residues of the N-terminus of a p53 protein or aportion thereof.

Even more typically, the antibody (or fragment thereof) has bindingaffinity for residues of the N-terminus of a p53 protein or a portionthereof. Yet more typically, the antibody (or fragment thereof) hasbinding affinity for residues about 10 to about 55. Still moretypically, the antibody (or fragment thereof) has binding affinity forresidues about 10 to about 25, or about 40 to about 50 or about 27 toabout 44 of the N-terminus of a p53 protein or portion thereof or about40 to about 44 of the N-terminus of a p53 protein or portion thereof.Even more typically, the antibody (or fragment thereof) has bindingaffinity for residues about 27 to about 44 of the N-terminus of a p53protein or a portion thereof. Still more typically, the antibody (orfragment thereof) has binding affinity for residues about 40 to about 44of the N-terminus of a p53 protein or a portion thereof.

Even more typically, the method is a method of locating a nucleotidesequence encoding a polypeptide of an antibody (or fragment thereof)having binding affinity to residues of the central domain of a p53protein or a portion is thereof.

Definitions

The term “antibody” means an immunoglobulin molecule able to bind to aspecific epitope on an antigen. Antibodies can be comprised of apolyclonal mixture, or may be monoclonal in nature. Further, antibodiescan be entire immunoglobulins derived from natural sources or fromrecombinant sources. The antibodies of the present invention may existin a variety of forms, including for example as a whole antibody, or asan antibody fragment, or other immunologically active fragment thereof,such as complementarity determining regions. Similarly, the antibody mayexist as an antibody fragment having functional antigen-binding domains,that is, heavy and light chain variable domains. Also, the antibodyfragment may exist in a form selected from the group consisting of: Fv,F_(ab), F(ab)₂, scFv (single chain Fv), dAb (single domain antibody),bi-specific antibodies, diabodies and triabodies.

By “antigen-recognising portion” is meant one or more portions of avariable region of an antibody (or fragment thereof) which areresponsible for binding and/or recognising the target antigen (orepitope or idiotype) of the antibody. For example, it includes the CDRregions or the whole variable region, or any combination of these tworegions including any changes in coding regions that may be induced inthe region without altering the binding properties of the antibody.

The antibody (or fragment thereof) of the present invention has bindingaffinity to a p53 protein or a portion thereof in vertebrates.Preferably, the antibody (or fragment thereof) of the present inventionhas binding affinity or avidity greater than about 10⁵ M⁻¹, morepreferably greater than about 10⁶ M⁻¹, more preferably still greaterthan about 10⁷ M⁻¹ and most preferably greater than about 10⁸ M⁻¹. Thetechniques for generating and reviewing binding affinity are reviewed inScatchard (1949), Annals of the New York Academy of Sciences, 51,660-672, and Munson (1983), Methods in Enzymology 92, 543-577, thecontents of each of which are incorporated herein by reference.

As used herein “gene transfer” means the process of introducing aforeign nucleic acid molecule into a cell. Gene transfer is commonlyperformed to enable the expression of a particular product encoded bythe gene. The product may include a protein, polypeptide, anti-sense DNAor RNA, or enzymatically active RNA. Gene transfer can be performed incultured cells or by direct administration into animals. Generally genetransfer involves the process of nucleic acid contact with a target cellby non-specific or receptor mediated interactions, uptake of nucleicacid into the cell through the membrane or by endocytosis, and releaseof nucleic acid into the cytoplasm from the plasma membrane or endosome.Expression may require, in addition, movement of the nucleic acid intothe nucleus of the cell and binding to appropriate nuclear factors fortranscription.

As used herein “gene therapy” is a form of gene transfer and is includedwithin the definition of gene transfer as used herein and specificallyrefers to gene transfer to express a therapeutic product from a cell invivo or in vitro. Gene transfer can be performed ex vivo on cells whichare then transplanted into a patient, or can be performed by directadministration of the nucleic acid or nucleic acid-protein complex intothe patient, or can be performed by transfer of modified cells into apatient.

As used herein, the term “naturally-occurring disease” refers to adisease that has spontaneously arisen, not iatragenically induced.

The term “wild-type”, in terms of a gene or a gene product, refers tothat gene or a gene product which is characteristic of most of themembers of a species occurring naturally, and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene or gene product.

The term “mutant”, in terms of a gene or gene product, refers a changein the gene or gene product when compared to the wild-type gene or geneproduct.

The term “isolated and purified” means that the material in question hasbeen removed from its host, and associated impurities reduced oreliminated. Essentially, it means an object species is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition) and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 30 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

The term “operably linked” refers to the situation wherein for example,a nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if iteffects the transcription of the coding sequence.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine valine, leucine, andisoleucine, a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Typically, conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

As used herein the term “polypeptide” means a polymer made up of aminoacids linked together by peptide bonds.

The term “analogue” as used herein with reference to a nucleic acidsequence means a sequence which is a derivative of the nucleic acidsequences of the invention, which derivative comprises addition,deletion, substitution of one or more bases and wherein the encodedpolypeptide retains substantially the same function as the polypeptideencoded by the nucleic acid sequences of SEQ ID NOS 1-30.

In the context of this specification, the term “comprising” means“including principally, but not necessarily solely”. Further, variationsof the word “comprising”, such as “comprise” and “comprises” havecorrespondingly varied meanings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The reactivity of Fab against varying concentrations of p53 in adirect ELISA. Fabs binding to p53 were detected with 9E10 followed by agoat anti-mouse specific HRP conjugated antibody. Anti-Tetanus Toxoidindicates the signal obtained when a Fab which reacted with Tetanustoxoid was used.

FIG. 2; Cross reactivity of Fabs with other antigens as assessed byELISA. Binding Fabs (from crude bacterial supernatant) were detectedwith 9E10 followed by a goat anti-mouse specific HRP conjugatedantibody. The signal obtained for p53 was four times greater thanobserved with other antigens.

FIG. 3:(A) Binding of Fab clones to recombinant p53 in bacteriallysates. The binding of DO7 was detected with an HRP-goat anti-mouse(lane 1). The human-anti-p53 Fabs (163.1, 5, 17.24 lanes 3-6) andhuman-anti-tetanus Fab (negative control, lane 2) were detected with9E10 followed by HRP-goat anti-mouse antibodies.

(B) Immunoblot analysis of immunoprecipitates from the colorectal cancercell line HT-29. Immunoprecipitation was performed using the DO7positive control antibody (lane 2), human Fab antibody reactive withtetanus toxoid (lane 1), Fab from clones 163.1, 5, 17, 24 (lanes 3-6respectively) and Protein A with lysate alone (lanes 7). Followingimmunoprecipitation and electroblotting the blots were incubated with agoat anti p53 antibody followed by a HRP-conjugated donkey anti-goatantibody.

FIG. 4: Deduced amino acid sequence of heavy (4A) and light chain (4B)clones reactive with p53. Replacement (uppercase) and silent mutations(lowercase) are shown with respect to the most homologous germlinesequence.

FIG. 5. Map of the mammalian expression vector. The heavy and lightchain genes of the selected Fab clones were cloned into the Not1 andSpe1 of pG1D105 and the Sal1 and Xba1 of pKN.101 respectively. The heavyand light chain vectors contain the neomycin resistance anddihydrofolate reductase genes, which confer resistance to theantibiotics G418 and methotrexate respectively and allow selection oftransformed cells.

FIG. 6: A 10% PAGE showing the purification of the whole anti-p53antibody from clone C4B4. The molecular weight markers are indicated onthe left of the gel, the arrows on the right show the position of theheavy and light chain.

FIG. 7: Cross-reactivity of C4B4 and DO7 with p53 and other antigens asassessed by ELISA. C4B4 was detected with a HRP conjugated goatanti-human specific antibody and DO7 with a HRP conjugated goatanti-mouse specific antibody. Reactivity with the HRP conjugated goatanti-human was used as a negative control.

FIG. 8: Immunoblot analysis of immunoprecipitates from the colorectalcancer cell line HT-29, MCF7 and MethA Immunoprecipitation was performedusing the purified C4B4 and the anti-p53 monoclonal antibody DO7. Aimmunoprecipitation with Protein A alone with lysate was used as anegative control. Following immunoprecipitation and electrobloting theblots were incubated with a goat anti-p53 antibody followed by a HRPconjugated donkey anti-goat-HRP. Immunoprecipitation with C4B4 resultedin a similar pattern of reactivity to that of the DO7. No significantreactivity was seen in the negative control.

FIG. 9: Alignment of the sequence of the p53 gene fragment peptides thatwere isolated. Three phage clones with unique nucleotide sequence werecompared to the sequence of p53 Fragment sizes ranged from 14-32 aminoacids and corresponded to a region in the amino terminus of p53.

FIG. 10: Immunohistochemical analysis of p53 staining in a colorectaltumour using the C4B4 antibody. Bound antibody was detected with HRPconjugated goat anti-human antibody. The larger frame shows positivenuclear staining in the colorectal epithelium (regions of brownstaining), while a similar region of tissue with no C4B4 is shown in thesmaller frame and does not demonstrate any nuclear staining of thetumour tissue (blue staining).

FIG. 11; The reactivity of varying concentration of 1159.8 against wholep53, the central domain of p53. Fab binding to p53 were detected with9E10 followed by alkaline phosphatase conjugated goat anti-mouseantibody. The reactivity of the antibody to BSA was used as a negativecontrol.

FIG. 12: Cross reactivity of Fabs with other antigens as assessed byELISA. Fab binding to p53 were detected with 9E10 followed by alkalinephosphatase conjugated goat anti-mouse antibody.

FIG. 13: Immunodetection of whole p53 and the central domain of p53using the Fabs 1159.8 and 163.1. A 4-20% SDS/PAGE was electroblottedonto PVDV and probed with the Fabs 1159.8 and 163.1, and the positivecontrol murine monoclonal antibodies, DO7 and Pab 240. Fabs weredetected with alkaline phosphatase conjugated goat anti-human Fab₂specific, while murine antibodies were detected with an with alkalinephosphatase conjugated goat anti-mouse antibody.

DETAILED DESCRIPTION OF THE INVENTION

1. Nucleic Acid Encoding a Polypeptide of an Antibody or fragmentthereof to p53

Included within the scope of this invention are the functionalequivalents of the herein-described isolated nucleic acid molecules. Thedegeneracy of the genetic code permits substitution of certain codons byother codons which specify the same amino acid and hence would give riseto the same protein. The nucleic acid sequence can vary substantiallysince, with the exception of methionine and tryptophan the known aminoacids can be coded for by more than one codon. Thus, portions or all ofthe polynucleotide sequences of the present invention could besynthesised to give a nucleic acid sequence significantly different fromthat described herein (FIG. 4). However the encoded amino acid sequencethereof would be preserved.

In addition, the nucleic acid sequence may comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end of the nucleic acidformula shown in FIG. 4, or a derivative thereof. For example, thepresent invention is intended to include any nucleic acid sequenceresulting from the addition of ATG as an initiation codon at the 5′-endof the inventive nucleic acid sequence or its derivative, or from theaddition of TTA. TAG or TGA as a termination codon at the 3′-end of theinventive nucleotide sequence or its derivative. Moreover, the nucleicacid molecule of the present invention may, as necessary, haverestriction endonuclease recognition sites added to its 5′-end and/or3′-end.

Such functional alterations of a given nucleic acid sequence afford anopportunity to promote secretion and/or processing of heterologousproteins encoded by foreign nucleic acid sequences fused thereto. Allvariations of the nucleotide sequences of the present invention, andfragments thereof permitted by the genetic code are, therefore, includedin this invention.

Further, it is possible to delete codons or to substitute one or morecodons by codons other than degenerate codons to produce a structurallymodified polypeptide, but one which has substantially the same utilityor activity of the polypeptide produced by the unmodified nucleic acidmolecule. As recognised in the art, the two polypeptides arefunctionally equivalent, as are the two nucleic acid molecules whichgive rise to their production, even though the differences between thenucleic acid molecules are not related to degeneracy of the geneticcode.

The nucleic acid molecule in accordance with the first or secondembodiments of the invention may also include an expression controlsequence operably linked to the coding sequences, includingnaturally-associated or heterologous promoter regions. Preferably, theexpression control sequences will be procaryotic in nature in vectorscapable of transforming or transfecting procaryotic host cells. Evenmore preferably, the polynucleotides encoding the antibodies of thepresent invention are cloned into a phage display vector.

However, the nucleic acid molecule in accordance with the first orsecond embodiments of the invention may also be cloned into a eucaryoticexpression system, and may also include an expression control sequenceoperably linked to the coding sequences, including naturally-associatedor heterologous promoter regions. Preferably, the expression controlsequences will be eucaryotic promoter systems in vectors capable oftransforming or transfecting eucaryotic host cells.

Once the vector has been incorporated into the appropriate host, thehost is maintained under conditions suitable for high level expressionof the nucleotide sequences and the collection and purification of theexpressed immunoglobulin.

Further the host cells are chosen such that upon insertion of the vectorinto the host selective features of the vector enable the relevantexpressed polypeptide to either be displayed on the surface of the hostcell, or secreted/expressed into the culture medium. Examples of suchcells include XLi-Blue and HB2151 respectively. Effectively, solubleexpression of Fab by any non-suppressor strain is envisaged, and thesemay include E. coli HB2151 or MC1061. Alternatively, a suppressor strainfor the expression of Fab fused to the surface of a phage, such as E.coli XL1-blue or TG-1α.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors will contain selection markers. Forexample, typical selection markers include ampicillin-resistance orhygromycin-resistance, thereby permitting detection of those cellstransformed with the desired DNA sequences.

In general, procaryotes can be used for cloning the DNA sequences of thepresent invention. E. coli is one procaryotic host particularly usefulfor cloning the DNA sequences of the present invention. Typically, E.coli produces antibody (or fragment thereof), such as Fab, by way of aphage particle which is itself produced in bacteria. Specific examplestrains include HB2151, which expresses a soluble antibody (or fragmentthereof), and XL1-Blue which expresses the antibody (or fragmentthereof) on the cell surface e.g., phage display.

Eucaryotic organisms, such as yeast are also useful for expression.Saccharomyces sp. is a preferred yeast host, with suitable vectorshaving expression control sequences, an origin of replication,termination sequences and the like as desired. Typical promoters include3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters include, among others, promoters from alcohol dehydrogenase 2,isocytochrome C, and enzymes responsible for maltose and galactoseutilisation.

Mammalian cells are also typical hosts for expressing nucleotidesegments encoding immunoglobulins or fragments thereof. A number ofsuitable host cell lines capable of secreting intact heterologousproteins have been developed in the art, and include CHO cell lines,various COS cell lines, HeLa cells, L cells and myeloma cell lines.Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, an enhancer,and necessary processing information sites, such as ribosome bindingsites, RNA splice sites, polyadenylation sites, and transcriptionalterminator sequences.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection andelectroporation are commonly utilised for procaryotic cells, whereascalcium phosphate treatment, electroporation, lipofection, biolistics orviral-based transfection may be used for other cellular hosts. Othermethods used to transform mammalian cells include the use oftransfection, transformation, conjugation protoplast fusion, polybrene,liposomes, electroporation, particle gun technology and microinjection(see, generally, Sambrook et al, 1989).

After introduction of the vector, recipient host cells are generallygrown in a selective medium, which inherently selects for the growth ofthose cells containing the introduced vector. A variety of incubationconditions can be used to form the polypeptides of the presentinvention, but the most preferred conditions are those which mimicphysiological.

Alternatively, modification or inactivation of the nucleic acidsequences of the invention may be undertaken using well known anti-sensetechniques, that is, involving a nucleotide sequence complementary tothe polypeptide encoding sequence. More specifically, production of apolypeptide encoded by the nucleic acids of the invention, may bealtered, that is, reduced or eliminated by introducing a polynucleotidesequencers) complementary to these nucleic acid sequences a encoding thepolypeptide(s) which may be transcribed in a cell, and is capable ofhybridising to the resulting mRNA produced in the cell. On the basisthat the reaction occurs conditions allowing the complementaryanti-sense nucleotide sequence to hybridise to the polypeptide mRNA, theamount of polypeptide translated is thus altered, that is, reduced oreliminated.

2. Polypeptide of an Antibody or Fragment thereof to p53 and/or Antibodyor Fragment thereof to p53.

Antibodies or immunoglobulins are typically composed of four covalentlybound peptide chains. For example an IgG antibody has two light chainsand two heavy chains. Each light chain is covalently bound to a heavychain. In turn each heavy chain is covalently linked to the other toform a “Y” configuration, also known as an immunoglobulin conformation.Fragments of these molecules, or even heavy or light chains alone, maybind antigen.

A normal antibody heavy or light chain has an N-terminal (NH₂) variable(V) regions and a C-terminal (COOH) constant (C) region. The heavy chainvariable region is referred to as V_(H) (including, for example, V_(γ)),and the light chain variable region is referred to as V_(L) (includingV_(κ) or V_(λ)). The variable region is the part of the molecule thatbinds to the antibody's cognate antigen, while the Fc region (the secondand third domains of the C region) on the heavy chain determines theantibody's effector function (e.g., complement fixation, opsonisation).Full-length immunoglobulin or antibody “light chains” are encoded by avariable region gene at the N-terminus and a κ (kappa) or λ (lambda)constant region gene at the COOH-terminus. Full-length immunoglobulin orantibody “heavy chains”, are similarly encoded by a variable region geneand one of the constant region genes, e.g., gamma. Typically, the“V_(L)” will include the portion of the light chain encoded by the V_(L)and J_(L) (J or joining region) gene segments and the “V_(H)” willinclude the portion of the heavy chain encoded by the V_(H), and D_(H)(D or diversity region) and J_(H) gene segments.

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions, alsocalled complementarity-determining regions or CDRs. The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three dimensionalspace. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs are typically referred to as CDR1, CDR2, and CDR3,numbered sequentially starting from the N-terminus.

The two types of light chains, κ (kappa) and λ (lambda), are referred toas isotypes. Isotypic determinants typically reside in the constantregion of the light chain, also referred to as the C_(L) in general, andC_(κ) or C_(λ) in particular. Likewise, the constant region of the heavychain molecule, also known as C_(H), determines the isotype of theantibody. Antibodies are referred to as IgM, IgD, IgG, IgA, and IgEdepending on the heavy chain isotype. The isotypes are encoded in the μ(mu), δ (delta), γ (gamma), α (alpha), and ε (epsilon) segments of theheavy chain constant region, respectively.

The heavy chain isotypes determine different effector functions of theantibody, such as opsonisation or complement fixation. In addition, theheavy chain isotype determines the secreted form of the antibody.Secreted IgG, IgD, and IgE isotypes are typically found in single unitor monomeric form. Secreted IgM isotype is found in pentameric form;secreted IgA can be found in both monomeric and dimeric form.

In a related aspect, the invention features a monoclonal antibody, or anFab, (Fab)₂, scFv (single chain Fv), dAb (single domain antibody),bi-specific antibodies, diabodies and triabodies, or otherimmunologically active fragment thereof (e.g., a CDR-region). Suchfragments are useful as immunosuppressive agents. Alternatively, theantibody of the invention may have attached to it an effector orreporter molecule. For instance, an antibody or fragment thereof of theinvention may have a macrocycle, for chelating a heavy metal atom, or atoxin, such as ricin, attached to it by a covalent bridging structure.In addition, the Fc fragment or CH₃ domain of a complete antibodymolecule may be replaced or conjugated by an enzyme or toxin molecule,such as chelates toxins, drugs or prodrugs, and a part of theimmunoglobulin chain may be bonded with a polypeptide effector orreporter molecule, such as biotin, fluorochromes, phosphatases andperoxidases. Bispecific antibodies may also be produced in accordancewith standard procedures well known to those skilled in the art.

The present invention further contemplates genetically modifying theantibody variable and/or constant regions to include effectivelyhomologous variable and constant region amino acid sequences. Generally,changes in the variable region will be made to improve or otherwisemodify antigen binding properties of the antibody or fragment thereof.Changes in the constant region will, in general, be made in order toimprove or otherwise modify biological properties, such as complementfixation, interaction with membranes, and other effector functions.

In the present context, effectively homologous refers to the conceptthat differences in the primary structure of the variable region of theantibody (or fragment thereof) may not alter the binding characteristicsof the antibody or fragment thereof. Changes of amino acids arepermissible in effectively homologous sequences so long as the resultantantibody or fragment thereof retains its desired property.

Amino acid changes in the polypeptide or the antibody or fragmentthereof may be effected by techniques well known persons skilled in therelevant art. For example, amino acid changes may be effected bynucleotide replacement techniques which include the addition, deletionor substitution of nucleotides, under the proviso that the properreading frame is maintained. Exemplary techniques include randommutagenesis, site-directed mutagenesis, oligonucleotide-mediated orpolynucleotide-mediated mutagenesis, deletion of selected region(s)through the use of existing or engineered restriction enzyme sites, andthe polymerase chain reaction.

In a related aspect, the invention further contemplates peptidefragments of the polypeptides of SEQ ID NOs 31-60. For example, peptidefragments comprising between about 5 and about 50 contiguous aminoacids, preferably between about 5 and about 35 amino acids, even morepreferably between about 5 and about 30 amino acids, even morepreferably still between about 5 and about 25 amino acids and yet morepreferably still between about 8 and about 20 amino acids arecontemplated in this aspect of the invention. It will be appreciated bythose skilled in art that such peptide fragments may or may not haveaffinity for a p53 protein or a portion thereof.

For example, the peptide fragment may be selected from the VL chainand/or from the VH chain of the polypeptides of the present invention.Preferably, the peptide fragment may be selected from thecomplementarity determining region (CDR) and/or from the frameworkregion (FR) of the VH and/or VL chain. More preferably still, thepeptide fragment is selected from the VH and/or VL region of the CDR.

The peptide fragments of the present invention find industrial use, forexample, in immunisation protocols to create an idiotypic response. Aswill be appreciated by those skilled in the art the peptide fragments ofthe present invention may be used to immunise a patient in need of suchimmunisation by administration of an amount effective to induce immunityto p53, either wild-type or mutant. One skilled in the art would beable, by routine experimentation, to determine what an effective,non-toxic amount of peptide fragment would be for the present purpose.Generally, however an effective dosage is expected to be in the range ofabout 5 milligrams to about 100 milligrams per dose, preferably about 5milligrams to about 75 milligrams per dose, more preferably about 10milligrams to about 50 miligrams per dose, even more preferably about 20milligrams to about 40 milligrams per dose.

The polypeptide of the third, fourth and fifth embodiment and/or theantibody of the sixth and seventh embodiments of the present inventionare also useful in functional studies of p53 protein in vertebrates. Aswill be apparent to those skilled in this art, example studies includeassays for determining p53 expression in normal and disease states, theeffect on cell growth and proliferation of antibody binding to p53. Forexample, functional studies may be performed in vivo or in vitro. Morepreferably, functional studies are performed in vitro.

3. Pharmaceutical/Therapeutic and Diagnostic Compositions

In another aspect, the invention features pharmaceutical compositions inwhich antibodies (or fragments thereof) of the present invention areprovided for therapeutic, prophylactic or diagnostic uses. Suchantibodies can also be provided as immunotoxins, that is, moleculeswhich are characterised by two components and are particularly usefulfor killing selected cells in vitro or in vivo. One component is acytotoxic agent which is usually fatal to a cell when attached orabsorbed. The second component, known as the “delivery vehicle” providesa means for delivering the toxic agent to a particular cell type, suchas carcinoma cells. The two components are commonly chemically bondedtogether by any of a variety of well-known chemical or geneticprocedures. For example, when the cytotoxic agent is a protein and thesecond component is an intact immunoglobulin, the linkage may be by wayof heterobifunctional crosslinkers, e.g., carbodiimide, glutaraldehyde,and the like.

Once expressed, polypeptides of the present invention can be purifiedaccording to standard procedures of the art, including HPLCpurification, size exclusion, ion-exchange and immuno-affinity (column)chromatography, gel electrophoresis and the like.

The antibodies of the present invention may be used as passive or activetherapeutic agents against a number of human diseases, including cancer,wherein such cancer may include: tumours of epithelial origin, such ascolo-rectal cancer, breast cancer, lung cancer, head and neck tumours,hepatic cancer, pancreatic cancer brain cancer, ovarian cancer, gastriccancer, bladder cancer, prostate cancer and urinary/genital tractcancer, oesophageal cancer, mesenchymal tumours such as sarcoma, andhaemopoietic tumours such as lymphoma.

The antibodies (or fragments thereof) of the present invention can beused either in their native form, or as part of ad antibody/chelate,antibody/drug, antibody/prodrug, antibodyltoxin or antibody/imagingmarker complex. Additionally, whole antibodies or antibody fragments(Fab₂, Fab, Fv) may be used as imaging reagents or as potential vaccinesor immunogens in active immunotherapy for the generation ofanti-idiotypic responses.

Conjugates of the antibody (or fragment thereof) and imaging marker(s)may be administered in a pharmaceutically effective amount for the invivo diagnostic assays of a number of human diseases, including cancer,wherein such cancer may include: tumours of epithelial origin, such ascobo-rectal cancer, breast cancer, lung cancer, head and neck tumours,hepatic cancer, pancreatic cancer, brain cancer, ovarian cancer, gastriccancer, bladder cancer, prostate cancer and urinary/genital tractcancer, oesophageat cancer, mesenchymal tumours, such as sarcoma; andhaemopoietic tumours, such as lymphoma, in a patient having a tumourthat expresses p53 and then detecting the presence of the imaging markerby appropriate detection means.

Administration and detection of the antibody/imaging marker, as well asmethods of conjugating the antibody/imaging marker, are accomplished bymethods readily known or readily determined in the art. The dosage ofsuch antibody/imaging marker will vary depending on the age and weightof the patient. Generally the dosage should be effective to visualise ordetect tumour sites, distinct from normal tissues. Preferably a one-timedosage will be between about 0.1 mg to about 200 mg. More preferably aone-time dosage will be between about 1 mg to about 150 mg, even morepreferably between about 5 mg to about 100 mg; even more preferablystill a one-time dosage will be between about 10 mg to about 50 mg.

Example imaging markers include substances which can be detected by agamma scanner or hand held gamma probe, and substances which can bedetected by nuclear magnetic resonance imaging using a nuclear magneticresonance spectrometer.

For example, the imaging marker which may be detected using a gammascanner include imaging markers selected from the group consisting of¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, and ^(99m)Tc.

An example of an imaging marker which can be detected using a nuclearmagnetic resonance spectrometer is gadolinium.

The amount of antibody (or fragment thereof) useful to produce atherapeutic effect can be determined by standard techniques well knownto those of ordinary skill in the art. The antibodies will generally beprovided by standard technique within a pharmaceutically acceptablebuffer and may be administered by any desired route. Because of theefficacy of the antibodies of the present invention, and their toleranceby humans it is possible to administer these antibodies repetitively inorder to combat various diseases/or disease states within a human.

One skilled in the art would be able by routine experimentation, todetermine what an effective, non-toxic amount of antibody (or fragmentthereof) would be for the purpose of inducing immunosuppression.Generally, however, an effective dosage is expected to be in the rangeof about 0.05 to about 100 milligrams per kilogram body weight per day,preferably about 0.05 to about 50 more preferably about 0.5 to about 25,even more preferably about 0.5 to about 10 milligrams per kilogram bodyweight per day. Alternatively, an effective dosage may be up to 500mg/m². Generally, an effective dosage is expected to be in the range ofabout 50 to about 500 mg/m², preferably about 50 to about 250 mg/m²,more preferably about 75 to about 250 mg/m², even more preferably about75 to about 150 mg/m².

The antibodies (or fragments thereof) of this invention should also beuseful for treating tumors in vertebrates. More specifically, theyshould be useful for reducing tumor size, inhibiting tumor growth and/orprolonging the survival time of tumor-bearing vertebrates.

Accordingly, this invention also relates to a method of treating tumorsin a human or other animal by administering to such human or animal aneffective, non-toxic amount of an antibody or fragment thereof. Oneskilled in the art would be able, by routine experimentation, todetermine what an effective non-toxic amount of antibody or fragmentthereof would be for the purpose of treating carcinogenic tumors.Generally, however an effective dosage is expected to be in the range ofabout 0.05 to about 100 milligrams per kilogram body weight per day,preferably about 0.05 to about 50, more preferably about 0.5 to about25, even more preferably about 0.5 about to about 10 milligrams perkilogram body weight per day. Alternatively, an effective dosage may beup to about 500 mg/m². Generally, an effective dosage is expected to bein the range of about 50 to about 500 mg/m², preferably about 50 toabout 250 mg/m² , more preferably about 75 to about 250 mg/m², even morepreferably about 75 to about 150 mg/m².

The antibodies of the invention may be administered to vertebrates, forexample humans or other animals in accordance with the above methods oftreatment in an amount sufficient to produce a therapeutic orprophylactic effect.

The antibodies of the invention can be administered to such human orother animal in a conventional dosage form prepared by combining theantibody or fragment thereof of the invention with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognised by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

The route of administration of the antibody (or fragment thereof) of theinvention may be oral, parenteral, by inhalation or topical. The termparenteral as used herein includes intravenous, intradermal,intramuscular, subcutaneous, rectal, vaginal or intraperitonealadministration. The subcutaneous and intramuscular forms of parenteraladministration are generally preferred.

The daily parenteral and oral dosage regimens for employing compounds ofthe invention to prophylactically or therapeutically induceimmunosuppression, or to therapeutically treat carcinogenic tumors willgenerally be in the range of about 0.05 to about 100, preferably about0.05 to about 50, more preferably about 0.5 to about 25, even morepreferably about 0.5 to about 10 milligrams per kilogram body weight perday. Alternatively, an effective dosage may be up to about 500 mg/m².Generally, an effective dosage is expected to be in the range of about50 to about 500 mg/m², preferably about 50 to about 250 mg/m², morepreferably about 75 to about 250 mg/m², even more preferably about 75 toabout 150 mg/m².

The antibody or fragment thereof of the invention may also beadministered by inhalation, that is, intranasal and/or oral inhalationadministration. Appropriate dosage forms for such administration, suchas an aerosol formulation or a metered dose inhaler, may be prepared byconventional techniques. The preferred dosage amount of a compound ofthe invention to be employed is generally within the range of about 0.05to about 100, preferably about 0.05 to about 50, more preferably about0.5 to about 25, even more preferably about 0.5 to about 10 milligramsper kilogram body weight per day. Alternatively, an effective dosage maybe up to about 500 mg/m². Generally, an effective dosage is expected tobe in the range of about 50 to about 500 mg/m², preferably about 50 toabout 250 mg/m², more preferably about 75 to about 250 mg/m², even morepreferably about 75 to about 150 mg/m².

The antibody or fragment thereof of the invention may also beadministered topically. By topical administration is meant non-systemicadministration and includes the application of an antibody (or, fragmentthereof) compound of the invention externally to the epidermis, to thebuccal cavity and instillation of such an antibody into the ear, eye andnose, and where it does not significantly enter the blood stream. Bysystemic administration is meant oral, intravenous, intraperitoneal andintramuscular administration. The amount of an antibody or fragmentthereof required for therapeutic or prophylactic effect will, of course,vary with the antibody chosen, the nature and severity of the conditionbeing treated and the animal undergoing treatment, and is ultimately atthe discretion of the physician. A suitable topical dose of an antibodyor fragment thereof of the invention will generally be within the rangeof about 1 to about 100 milligrams per kilogram body weight daily,preferably about 0.05 to about 50, more preferably about 0.5 to about 25even more preferably about 0.5 to about 10 milligrams per kilogram bodyweight per day. Alternatively, an effective dosage may be up to about500 mg/m². Generally, an effective dosage is expected to be in the rangeof about 50 to about 500 mg/m², preferably about 50 to about 250 mg/m²,more preferably about 75 to about 250 mg/m², even more preferably about75 to about 150 mg/m².

In the administration of therapeutic formulations in accordance with thepresent invention and herein disclosed, there are preferred non-toxicpharmaceutical carriers diluents, excipients and/or adjuvants. Foradministration of the above formulations the polypeptides to be used areadmixed with these non-toxic carriers, diluents, excipients and/oradjuvants and may be in the form of capsules, aqueous or oilysuspensions, emulsions, syrups, elixirs or injectable solutions.

Examples of pharmaceutically and veterinarily acceptable carriers ordiluents are demineralised or distilled water; saline solution;vegetable based oils such as peanut oil, safflower oil, olive oil,cottonseed oil, maize oil, sesame oils such as peanut oil, saffloweroil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil orcoconut oil, silicone oils, including polysiloxanes, such as methylpolysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane;volatile silicones; mineral oils such as liquid paraffin, soft paraffinor squalane; cellulose derivatives such as methyl cellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose orhydroxypropylmethylcellulose; lower alkanols, for example ethanol oriso-propanol, lower aralkanols; lower polyalkylene glycols or loweralkylene glycols, for example polyethylene glycol, polypropylene glycol,ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin:fatty acid esters such as isopropyl palmitate, isopropyl myristate orethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth orgum acacia, and petroleum jelly. Typically, the carrier or carriers willform from 10% to 99.9% by weight of the compositions.

Some examples of suitable carriers, diluents, excipients and adjuvantsfor oral use include peanut oil, liquid paraffin, sodiumcarboxymethylcellulose, methylcellulose, sodium alginate, gum acacia,gum tragacanth, dextrose, sucrose, sorbitol mannitol, gelatine andlecithin. In addition these oral formulations may contain suitableflavouring and colourings agents. When used in capsule form the capsulesmay be coated with compounds such as glyceryl monostearate or glyceryldistearate which delay disintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents,preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable inhuman and veterinary pharmaceutical practice sweeteners, disintegratingagents, diluents flavourings, coating agents, preservatives, lubricantsand/or time delay agents. Suitable binders include gum acacia, gelatine,corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose orpolyethylene glycol. Suitable sweeteners include sucrose, lactose,glucose, aspartame or saccharine. Suitable disintegrating agents includecorn starch methylcellulose, polyvinylpyrrolidone guar gum, xanthan gumbentonite, alginic acid or agar. Suitable diluents include lactose,sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate,calcium silicate or dicalcium phosphate. Suitable flavouring agentsinclude peppermint oil, oil of wintergreen, cherry, orange or raspberryflavouring. Suitable coating agents include polymers or copolymers ofacrylic acid and/or methacrylic acid and/or their esters, waxes, fattyalcohols zein, shellac or gluten. Suitable preservatives include sodiumbenzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben,propyl paraben or sodium bisulphite. Suitable lubricants includemagnesium stearate, stearic acid, sodium oleate, sodium chloride ortalc. Suitable time delay agents include glyceryl monostearate orglyceryl distearate.

Liquid forms for oral administration may contain, in addition to theabove agents, a liquid carrier. Suitable liquid carriers include water,oils such as olive oil, peanut oil, sesame oil, sunflower oil, saffloweroil, arachis oil, coconut oil, liquid paraffin, ethylene glycol,propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol,glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further comprise dispersingagents and/or suspending agents Suitable suspending agents includesodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginateor acetyl alcohol. Suitable dispersing agents include lecithin,polyoxyethylene esters of fatty acids such as stearic acidpolyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate,polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate andthe like.

The emulsions for oral administration may further comprise one or moreemulsifying agents. Suitable emulsifying agents include dispersingagents as exemplified above or natural gums such as guar gum, gum acaciaor gum tragacanth.

For administration as an injectable solution or suspension, non-toxicparenterally acceptable diluents or carriers can include, Ringer'ssolution, isotonic saline, phosphate buffered saline, ethanol and 1,2propylene glycol.

Further, a vaccine composition containing the recombinant polypeptidemay be prepared for use by standard methods, well known to those ofordinary skill in the art. In one embodiment, the immunogenic peptidemay be produced in a recombinant system by expression of thepolynucleotide sequence (or a fragment thereof) in accordance with thepresent invention, and subsequently isolated. For example, microbialcells containing the exogenous gene of interest may be cultured in largevolume bioreactors, then collected by centrifugation and subsequentlyruptured, for instance by high pressure homogenisation. The resultingcell lysate may be resuspended in appropriate diluent such as thosedescribed herein, and filtered to obtain an aqueous suspension of theimmunogen. The recombinant protein can be administered in crude form,for example, by diluting in a 0.1M phosphate buffer (pH 7.4) to 50-500μg/ml concentration, and then passing through a sterile 0.22 micronfilter.

Alternatively, a vaccine composition containing the recombinantpolypeptide may be prepared in a mammalian expression system, utilisinghost cells such as Chinese Hamster Ovary (CHO) cells. The antibody (orfragment thereof) having binding affinity to p53 or a portion thereofmay be manufactured using batch fermentation with serum free medium.After fermentation the antibody may be purified via a multistepprocedure incorporating chromatography and viral inactivation/removalsteps. For instance, the antibody may be first separated by Protein Aaffinity chromatography and then treated with solvent/detergent toinactivate any lipid enveloped viruses. Further purification, typicallyby anion and cation exchange chromatography may be used to removeresidual proteins, solvents/detergents and nucleic acids. The purifiedantibody may be further purified and formulated into 0.9% saline usinggel filtration columns. The formulated bulk preparation may then besterilised and viral filtered and dispensed.

Alternatively, the antibody (or a fragment thereof) of the presentinvention may be used as an idiotypic immunogen. As is known to those ofskill in the art, when used in this manner, the antibody (or a fragmentthereof) of the present invention may function as an immunogen andelicit a second antibody (Ab2) and T cell (T₂) response againstidiotopes of the original antibody (Ab1). Ab2 antibodies can bind toepitopes on the original antibody including the antigen binding site(idiotype). The anti-idiotypic antibody, Ab2, can spontaneously induceanti-anti-idiotypic antibodies (Ab3) as well as T cells (T₃) which mayrecognise the same epitope as Ab1. Since the first antibody binds boththe p53 epitope and Ab2, Ab2 mimics the structure of the antigenicepitope (on p53). A proportion of Ab3 antibodies bind to the sameepitope as the original antibody (Ab1), and may augment and prolong theefficacy of the original antibody. Induction of this anti-idiotypicnetwork results in protection from metastases partly through theinduction of p53-specific CTLs.

Alternatively, a vaccine composition containing a peptide fragment ofthe polypeptide of the present invention may be prepared by synthesis ofa peptide, using standard methods known to those in the art, such as byautomated synthesis on, for instance, an Applied Biosystems model 430A.For example, the peptide may comprise selected amino acid regions of theCDR and/or FR of the polypeptide of the invention. The synthetic peptidecan be administered for example, after diluting in a 0.1M phosphatebuffer (pH 7.4) to 50-500 μg/ml concentration, and passing through asterile 0.22 micron filter.

Alternatively, the vaccine may be a DNA based vaccine. In one aspect,the DNA based vaccine may comprise naked DNA comprising a nucleic acidmolecule as defined in the first or second embodiments of the invention,or a fragment thereof.

In another aspect, the DNA based vaccine may comprise a nucleic acidmolecule as defined in the first or second embodiments of the invention,or a fragment thereof, cloned into an expression vector. Typically, theexpression vector is a eucaryotic expression vector and may includeexpression control sequences, such as an origin of replication apromoter, an enhancer and necessary processing information sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences.

A typical vaccination regime is to deliver the vaccine in multiple dosesgenerally one, two or three equal doses.

In general to induce the production of antibodies to the vaccines of theinvention they can be oteogenous or aqueous suspensions formulated inaccordance with known methods in the art using suitable dispersing,suspension and/or wetting agents. Examples of suitable dispersing,suspension and wetting agents include Freund's complete/incompleteadjuvant, Montenide Marcol adjuvant and phosphate buffered saline, andmannan.

It will be appreciated that the examples referred to above areillustrative only and other suitable carriers, diluents, excipients andadjuvants known to the art may be employed without departing from thespirit of the invention.

4. An Antibody/Nucleic Acid Based Method and Kit for Detecting p53

The present invention also encompasses a method of detecting a p53polypeptide in a sample, wherein the method comprises:

(a) contacting a sample with the antibody (or fragment thereof) asdefined in accordance with the sixth or seventh embodiments of theinvention, and

(b) detecting the presence of the antibody (or fragment thereof) boundto a p53 polypeptide.

Typically, altered levels of p53 polypeptide may indicate the presenceor onset of disease, wherein an example of such a disease is cancer.

Conditions for incubating an antibody (or fragment thereof) with a testsample vary widely, depending on the format of detection used in theassay the detection method, and the type and nature of the antibodyused. A person of ordinary skill in the art would readily appreciatethat any one of the commonly available immunological assays could beused in performing the method of as detection. For example these assaysinclude radioimmunoassays, enzyme-linked immunosorbent assays and/orimmunoflourescent assays.

Further, the test sample used in the assay may consist of tissue, cells,protein or membrane extracts of cells, and biological fluids such asblood, serum, plasma or urine.

A kit for performing the above method of the invention contains all thenecessary reagents to carry out the above methods of detection. Forexample, the kit may comprise the following containers:

(a) a first container containing the antibody (or fragment thereof) ofthe present invention,

(b) a second container containing a conjugate comprising a bindingpartner of the antibody (or fragment thereof), together with adetectable label.

Typically, the kit may further comprise one or more other containers,containing other components, such as wash reagents and other reagentscapable of detecting the presence of bound antibodies. More typically,the detection reagents may include, labelled (secondary) antibodies, orwhere the antibody (or fragment thereof) of the present invention isitself labelled, the compartments may comprise antibody binding reagentscapable of reacting with the labelled antibody (or fragment thereof) ofthe present invention.

Further, the kit of the present invention, as described above inrelation to antibodies, can be readily incorporated, without theexpenditure of inventive ingenuity into a kit for nucleic acid probes.One skilled in the art would select the nucleic acid probe from thepolynucleotides of the present invention, according to techniques knownin the art as described above. Samples to be tested include but shouldnot be limited to RNA samples of human tissue.

Such a kit comprises at least one container means having disposedtherein the above-described nucleic acid probe. The kit may furthercomprise other containers comprising one or more of the following: washreagents and reagents capable of detecting the presence of bound nucleicacid probe. Examples of detection reagents include, but are not limitedto radiolabelled probes, enzymatic labelled probes (horseradishperoxidase, alkaline phosphatase), and affinity labelled probes (biotin,avidin, or steptavidin).

In detail, a compartmentalised kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer of reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the probe or primers used in the assay,containers which contain wash reagents (such as phosphate bufferedsaline Tris-buffers, and like), and containers which contain the reagentdetect the hybridised probe, bound antibody amide product, or the like.

Furthermore, one skilled in the art would readily recognise that thenucleic acid probes in the present invention can readily be incorporatedinto one of the established kit formats which are known in the art.

5. Gene Therapy

In its simplest form, gene transfer can be performed by simply injectingminute amounts of DNA into the nucleus of a cell through a process ofmicroinjection. Once recombinant genes are introduced into a cell, theycan be recognised by the cells normal mechanisms for transcription andtranslation, and a gene produce will be expressed.

Other methods have also been attempted for introducing DNA into largernumbers of cells. These methods include: transfection, wherein DNA isprecipitated with CaPO₄ and taken into cells by pinocytosis;electroporation, wherein cells are exposed to large voltage pulses tointroduce holes into the membrane, lipofection/liposome fusion, whereinDNA is packaged into lipophilic vesicles which fuse with a target cell;and particle bombardment using DNA bound to small projectiles. Anothermethod for introducing DNA into cells is to couple the DNA to chemicallymodified proteins.

In one embodiment, an expression vector containing the polynucleotidesequence according to the present invention is inserted into cells, thecells are grown in vitro and then infused in large numbers intopatients. Expression vectors derived from viruses such as retroviruses,vaccinia virus, adenovirus, adeno-associated virus, herpes viruses,several RNA viruses, or bovine papilloma virus, may be used for deliveryof the polynucleotide sequences of the invention into the targeted cellpopulation (e.g., tumour cells). Methods which are well known to thoseskilled in the art can be used to construct recombinant viral vectorscontaining coding sequences. Alternatively, recombinant nucleic acidmolecules encoding protein sequences can be used as naked DNA or inreconstituted system, for example, liposomes or other lipid systems fordelivery to target cells.

It has also been shown that adenovirus proteins are capable ofdestabilising endosomes and enhancing the uptake of DNA into cells. Theadmixture of adenovirus to solutions containing DNA complexes, or thebinding of DNA to polylysine covalently attached to adenovirus usingprotein crosslinking agents substantially improves the uptake andexpression of the recombinant gene.

The invention will now be described in greater detail by reference tospecific Examples, which should not be construed as in any way limitingon the scope thereof.

EXAMPLES Example 1 Isolation and Characterisation of Anti-p53 HumanAntibodies Materials & Methods Patient Data

After obtaining informed consent, blood and tissue samples werecollected from 100 individuals seen at St Vincent's Hospital from1993-1997 who were undergoing resection of colorectal cancer.

Clotted blood was centrifuged at 2000 g for 10 min and serum stored inaliquots at −70° C. prior to use. Samples from 50 healthy individualswere obtained and used as controls in all ELISA and immunoprecipitationexperiments. A fresh pericolic lymph node in the region of the tumourwas harvested from colectomy tissue and frozen in liquid nitrogen priorto RNA extraction (6).

Immunohistochemical Detection of p53

Sections of paraffin embedded tumour tissue from each individual weresubjected to immunohistochemical analysis of p53 as previously described(7). Tumour tissue was considered to have accumulated mutant p53 whenthe average of ten high powered fields showed greater than 5% of tumorcells with nuclear staining, in the absence of staining in the stromalcells and normal epithelium.

Production of Recombinant p53

Recombinant p53 was expressed and purified. Briefly, a cDNA clone ofwild type p53 in the expression vector pET19b was transfected into E.coli strain BL-21(DE3) (Novagene Inc. Madison, Wis.). Protein waspurified from crude bacterial lysates using Ni₂ ⁺ resin. p53 purity wasassessed by polyacrylamide gel electrophoresis (PAGE) and thenimmunoblotting. The protein concentration was determined using theBiochonincic acid method with reference to a standard curve generatedwith bovine serum albumin (BSA).

Detection of Anti-p53 Serum Antibodies

Wells of a microtitre plate (Polysorb, Nunc Denmark) were coated withpurified recombinant p53 (5 μg/ml in Phosphate Buffered Saline PBS)overnight at 4° C. Coated wells were washed three times each with 200 μlof PBS and then blocked with PBS/2% BSA for 1 hour at room temperature(RT) Patient serum samples (n=100) were diluted 1 in 100 in PBS and thenapplied in duplicate to the p53 and incubated for 1 hour at RT. Bindingantibodies were detected with an alkaline phosphatase conjugated goatanti-human IgG Fc-specific antibody (0.5 μg/ml in PBS/2% BSA. JacksonImmuno Research Lab Inc, PA, USA). The reactivity of each patient to p53was expressed as a value relative to a standard curve generated fromcontrol serum known to contain anti-p53 antibodies, as describedpreviously (7). Serum activity was compared to a healthy group ofvolunteers (n=50) and considered positive for anti-p53 antibodies whenthe anti-p53 score was >2 standard deviations above the mean of thenormal group.

The isotype of antibodies in reactive sera was assessed using the aboveprotocol, except, that the anti-human IgG Fc-specific antibody wasreplaced with mouse anti-human IgG (IgG1, IgG2, IgG3, and IgG4: DAKOCorp CA, USA) isotype specific antibody (1 μg/ml) and detected with analkaline phosphatase (AP) conjugated goat anti-mouse antibody (0.5 μg/mlin PBS/2% BSA: Jackson Immuno Research Lab Inc).

The anti-p53 serum titre was defined as the lowest dilution of serumthat generated a signal of 3 times above background.

Library Construction and Biopanning

Pericolic lymph nodes were ground to a fine powder in liquid nitrogen,and total RNA extracted using standard procedures (8). IgG1 kappa chainFab libraries were constructed in the MCO1 vector as describedpreviously (9). Briefly, immunoglobulin genes were amplified by RT-PCRusing primers specific for human kappa and IgG1 immunoglobulin genesfollowed by digestion with Sac1/Xba1 or Spe1/Xho1 respectively. Theproducts were then cloned sequentially (light chain then heavy chain)into the phage display phagemid vector. MCO1, and the combinatoriallibraries electroporated into XL1-blue cells and packaged with helperphage to give the primary antibody phage library.

The size of the library was calculated from a proportion of clones takenafter electroporation (n=20 for each library) of the final heavy andlight chain construct. A diagnostic PCR amplifying the variable regionof the heavy and light chain and BstN1 finger printing (see below) wereused to calculate the number of clones with unique heavy and light chaininserts. On this basis the total library size was estimated.

Wells of a microtitre plate were coated with recombinant p53 asdescribed above, and then washed with PBS and blocked with BSA (2% v/v)/PBS. Aliquots of the phage antibody libraries (10¹² cfu in 100 μl) wereapplied to each well and incubated at room temperature for two hours.Excess phage were washed from the plate with six washes with PBS/Tweenfollowed by two washes in PBS. Adherent phage were then eluted with 100μl of 0.1 M glycine pH 3.0 for 10 min at room temperature, andneutralised with 1 M Tris pH 8. Eluted phage were reamplified for thenext round of panning as described previously (6). The panning procedurewas carried out five times. An aliquot was taken from the eluted outputfrom each round of panning and used to infect the E. coli non suppressorstrain HB2151 for the production of soluble Fab. Infected bacteria wereplated onto Luria Broth agar with 50 μg/ml of carbenicillin and singlecolonies were picked for soluble Fab production.

Analysis of Soluble Fab Reactivity by ELISA

Cultures were grown overnight from a single colony at 37° C. in 2YTbroth with 2% glucose (v/v) and 50 μg/ml of carbenicillin(2YT/glu/carb). These were then diluted 1 in 100 in 2YT/glu/carb andgrown at 37° C. until an OD of 0.8. The cultures were then centrifugedand resuspended in 2YT containing 1M IPTG and 50 μg/ml of carbenicillinand grown overnight at 30° C. Following centrifugation, the supernatantfrom the overnight cultures was assessed for anti-p53 Fabs by ELISA.

Culture supernatant was applied in duplicate to ELISA plates coated withp53, and incubated for 2 hours at RT. After washing with PBS, 100 μl ofthe anti-myc monoclonal antibody, 9E10 was added to each well (detectingthe myc tag on the C terminus of the heavy chain 0.5 μg/ml in PBS/0.5%BSA) and incubated at room temperature for 1 hour. The wells were againwashed and HRP conjugated goat anti-mouse (0.5 μg/ml in PBS/2% BSA:Jackson Immuno Research Lab Inc) antibody was added. After furtherwashing, colour was developed with 100 μl of TMB substrate (Kirkegaardand Perry Laboratories, Gaithersburg Md.) and the reaction was stoppedwith 50 μl of 1 M H₂SO₄. Clones were considered positive where the ODwas more than three times the signal seen in wells not coated with p53.In each ELISA, a negative control without 9E10 was used to detect crossreactivity of Fab, secondary antibodies and p53.

Reactive anti-p53 Fabs were reanalysed using p53 coated atconcentrations from 10-0.015 μg/ml, or by incubating Fab with 50 μg/mlof soluble p53 for 1 hr prior to application on the p53 coated ELISAplate (1 μg/ml). To confirm that the light chain was involved in bindingto p53 the ELISA was repeated using a biotinylated goat anti-human kappaspecific antibody (0.2 μg/ml in PBS/2% BSA, Rockland, Gilbertsville,Pa.) followed by HRP conjugated streptavidin (0.05 μg/ml in PBS/2% BSA,DAKO Corp).

The cross reactivity of Fabs with other antigens was assessed by ELISAusing a similar method to that described for p53. The following antigensand concentrations were used, insulin (5 μg/ml). ErbB2 extracellulardomain (5 μg/ml; gift from Ruth Lyons, Garvan Institute Sydney,Australia). Muc1 (5 μg/ml, gift Dr. Ian McKenzie, Austin ResearchInstitute, Melbourne, Australia) and CEA (5 μg/ml, extracted from tissueas described by Matsuoka et al 1991), tetanus toxoid (1 μg/ml; CSL,Melbourne, Australia), BSA (1 μg/ml: Sigma-Aldrich. Castle Hill,Australia) and keyhole limpet haemocyanin (1 μg/ml, Sigma-Aldrich).

Analysis of Fab Reactivity by Immunoprecipitation

The colorectal cancer cell line HT29, which contains mutant p53, wasused to assess the reactivity of Fabs with human p53 from eucaryoticcells. Approximately 10⁷ cells were lysed in TNES buffer (50 mM Tris pH7.5, 2 mM EDTA, 100 mM NaCl, 1% NP-40, protease inhibitor cocktail[Boehringer Mannheim, Castle Hill, Australia] and 1 mM PMSF) and thencell debris removed by centrifugation at 10000 g for 10 min.Approximately 250 μg of the lysate was used in each immunoprecipitation.Either the mouse anti DO7 (0-5 μg; DAKO Corp) or the bacteriallyexpressed Fab was added to the lysate and incubated for 1 hour at 4° C.The anti-myc 9E10 antibody (1 μg) was then added to the mixturecontaining Fab and incubated for 1 hour at 4° C. At this point 20 μl of(packed volume) protein A-sepharose (Zymed Laboratories Inc. SanFrancisco, Calif.) was added to all tubes and incubated for a further 1hour at 4° C. The protein A sepharose was washed four times with PBS,and subject to 10% PAGE under denaturing and reduced conditions.Proteins were transferred to PVDF membrane by electroblotting, blockedwith 10% skim milk powder and probed with a goat anti-p53 antibodyspecific for the N terminal region of the protein (Santa Cruz Biotech,Santa Cruz, Calif.). This was followed by a donkey anti-goat-HRPantibody (Jackson Immuno Research Lab Inc), and then the blots weredeveloped using chemiluminescent substrate (DuPont NEN, North Sydney,Australia). A negative Fab control (Fab specific for tetanus toxoid),and a Protein A sepharose and extract only control, were included ineach experiment as negative controls.

Epitope Mapping

A set of deletion mutants derived from human p53 were used in epitopemapping. The deletion mutants used were Hup53, 3M (residues 1-393), 3R(1-223), 4U (1-106), 11 (27-393) and 18 (44-393) as described by (10).Briefly cultures of E. coli (BS21 DE3λ) containing the constructs weregrow to OD 0.8. The cells were lysed in bacterial lysis buffer (50 mMTris pH 7.5, 10 mM EDTA, 50 mM NaCl, 1% NP-40 and 1 mM PMSF) and 50 μlof the lysate were subject to PAGE and electroblotting as describedabove. Bacterial expressed Fab was incubated with the membrane for 1hour at RT and then washed with PBS. Bound Fab was detected with 9E10and HRP-conjugated goat anti-mouse. Negative controls were as describedabove.

Sequence Analysis

The variable region of selected clones was sequenced using a cyclesequencing kit according to the manufacturers specifications (Promega,Madison, Wis.) Miniprep DNA was prepared by alkaline lysis and bothstrands of DNA sequenced using primers outside the variable region. Theprimers used for sequencing the light chain were 5′-AA GAC AGC TAT CGCGAT T (OmpA leader sequence) and 5′-ATG AAG ACA GAT GGT GCA GC (5′ endof the kappa constant region) and the heavy chain 5′-CTA CGG CAG CCG CTGGAT TG (PelB leader sequence) and 5′-GGA AGT AGT CCT TGA CCA G (5′ endof the IgG CH1 region).

The heavy and light chain variable region for Fab clones was matched toavailable V genes, D genes and J genes using the DNA plot alignmentpackage and V base sequence data base.

Using the method of Chang and Casali (11), the frequency of replacementmutations (R) in the CDR and framework (FR), for each of the p53antibodies, was calculated with respect to its closest germ line gene.The probability that replacement mutations were occurring at a frequencyabove or below the expected random frequency was calculated in abinomial distribution model, using the expected number of R mutations inthe germline gene, the actual number of observed R mutations in the Fabsequences, and the probability of R mutations localising to the CDR orFRs (11). Amino acids from 1-94 of the heavy chain and 1-95 for thelight chain were used for the analysis of R mutations. Amino acidresidues occurring as a result of primer sequence in the FR1 region wereexcluded from the analysis. A p value of less than 0.05 indicated thatthe R mutations had occurred in a non-random fashion.

Fab Purification

Soluble Fab was precipitated with ammonium sulphate (35% (w/v) final),resuspended in 5 ml of PBS and then purified by IMAC affinitychromatography. Eluted fractions containing Fab were pooled and thenfractionated by size exclusion chromatography (Superdex 200 Pharmacia)in HBS buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA and 0.005% NP40). Purity was assessed by PAGE and silver staining.

BIAcore Analysis of Selected Fabs

Recombinant p53 was coupled to a CM5 chip using standard amineimmobilisation protocols. The chip was activated using 50 mMN-hydroxysuccinimide and 200 mMN-(dimethylaminopropyl)-N′-ethylcarbodimide. Recombinant p53 at 100μg/ml in PBS diluted 1 in 10 in sodium acetate (1 M, pH 4.8) wasinjected at a flow rate of 10 μl/min. No greater than 400 RU werecoupled to the chip for affinity analysis.

All measurements were carried out in HBS buffer. For the analysis ofaffinity, concentrations of Fab ranging from 10-200 nM were injected for90 sec at a flow rate of 30 μl/min over 2 flow cells one with coupledP53 and the other without. Dissociation was measured over 90 sec by theinjection of HBS buffer. The chip was regenerated with 20 μl of 1 Mglycine pH 2 at 30 μl/min flow rate. The RU of the blank flow cell wassubtracted from the p53 coupled cell and the affinity constantscalculated using the BIAevaluation 3 software package for a global fit.

Results Patient Serum Analysis and Antibody Library Construction

Of the 100 patients with colorectal cancer screened for antibodiesagainst p53. 17 were found to have anti-p53 antibodies. From thepatients found to have p53 reactive serum six were selected for furtherstudy including one patient with no detectable anti-p53 antibodies as anegative control. In addition, each of the patients was assessed for thepredominant IgG isotype reactive with p53. It was found that all theindividuals selected had predominantly IgG1 reactive anti-p53antibodies. IgG1k antibody libraries were therefore constructed from thepericolic lymph node tissue taken from these six colorectal cancerpatients. The size of the antibody libraries from each of theconstructed individuals, together with clinical data, serum andreactivity against full length p53 is shown in Table 1.

Anti-p53 Fab Selection

Each antibody library was subjected to five rounds of panning againstrecombinant p53. A 20-100 fold increase in the number of eluted phagewere observed in rounds 4 and 5.

No Fabs with reactivity against p53 were identified from 32 phage clonesisolated from each library after each of the first three rounds ofpanning (total number of clones analysed=960). The library from patient163 was found to have 1/32 p53 reactive clones from round 4 and 42/128p53 reactive clones from round five. No positive clones from rounds fouror five (96 clones analysed from output phage) were identified frompatient antibody libraries 100, 107, 149, 357 or 790 (192 phage clonesanalysed from each library).

The 43 p53 reactive clones isolated from library 163 were analysed byrestriction enzyme digestion and five clones were eliminated fromfurther analysis on the basis of lacking a heavy chain of the correctsize. All clones had light chain inserts of the expected size. Theremaining 38 clones were DNA fingerprinted from variable region PCRproducts using the frequent cutting restriction enzyme BstN1. Thisallowed the identification of four unique heavy chain BstN1 profileswhich paired with five unique light chain profiles giving a total of 14clones with unique heavy and light chain combinations (results notshown). Four clones with unique heavy chain were epitope mapped andanalysed for reactivity against recombinant p53, cell line derived p53as well as for cross reactivity with other antigens (clones 163.1,163.5, 163.17, 163.24). The nucleotide sequence of the 14 clones withunique heavy and light chain combinations was determined, the deducedamino acid sequence generated and the mutation pattern analysed.

Conformation of Anti-p53 Fab Reactivity

The reactivity of clones 163.1, 5, 17 and 24 with varying concentrationsof p53 is shown in FIG. 1. The reactivity of the Fabs against p53 wasalso demonstrated using the sheep anti-human kappa antibody (results notshown) involvement of both heavy and light chain in p53 binding. Whenpre-incubated with excess p53 prior to ELISA analysis the signal wasreduced to between 11-27% of the levels observed in the standardprotocol (results not shown). Furthermore, the four clones showed noreactivity against other antigens, including, CEA, erbB2, MUC-1,insulin, tetanus toxoid, KLH and BSA (FIG. 2).

The ability of the Fabs to detect p53 in bacterial lysates was assessedby Western analysis (FIG. 3 a). The Fabs were able to detect p53 in thelysate but didn't appear to react with other proteins. In addition itwas found that each of the Fabs were able to immunoprecipitate mutantp53 from the human colorectal cancer cell line HT-29 (FIG. 3 b).

Epitope Mapping

Epitope mapping of the Fab clones 163.1, 5, 17 and 24 showed that allwere reactive with full length Hup53, and deletion constructs 3M, 3R, 4Uand 11. None of the clones were reactive with the 18 construct (residues44-393), indicating that the Fabs were reactive with an epitope betweenresidues 27 and 44 (inclusive) of human p53.

Affinity Analysis

The dissociation constants for the antibodies 163.1, 5, 17 and 24 were1.19*10⁻⁸, 1.5*10⁻⁸, 1.57*10⁻⁸ and 1.38*10⁻⁸ respectively. The χ² valuewere all less than 1 when using the model for 1:1 interaction with adrifting baseline.

Sequence Analysis

For each of the 14 clones the closest germ line gene match and thepercent nucleotide difference from this gene is shown in Table II. Acomparison of the variable region of the 14 Fab clones showed that allthe clones had greater than 95% homology with each other and appeared tohave the same V gene D gene and J gene combination (Table II). The Vregion of these clones consisted of the V gene DP-7 (VH1-46) from theVH1 gene family, and the J gene. JH 4b No D segment gene could beassigned to these clones with confidence due to the lack of homologywith known D gene sequences, although all clones had a similar Dregions. All the heavy chains of these 14 clones had extensive mutationsthroughout the V gene region. The percentage difference between theheavy chain V gene and the matched germ line V gene ranged from14.6-18.5%. The mutations were frequent not only in the COR regions butalso throughout FR1 and FR3 regions. There was relatively few mutationsin the FR2 region. The light chain partners of these 14 clones hadgreater homology with the matched V gene than the heavy chain, with thepercentage of mutations ranging from 0-5.9%. The light chain partners ofthese clones used the same light chain V gene DPK-24 in combination witheither the JK2 or JK4 gene.

Mutation Analysis

The deduced amino acid sequences from the 14 clones was used to deducethe replacement and silent mutations within FR and CDR regions (FIG. 4),and these values used to calculate the probability that replacementmutations in FR or CDR were not random. Random mutations, eitherreplacement or silent, occur evenly throughout a given sequence, whileantigen driven responses are often localised and result in a higher orlower proportion of replacement mutations depending on the selectionpressures defined by antigen selection (11). The probability that themutations in the FR and CDR regions arose as a result of antigen drivenselection is shown in Table III.

All the heavy chains derived from the UP-7 V gene, had p values lessthan 0.05 as a result of a lower proportion of replacement mutations inthe FR than was expected. This suggests antigen-driven B cell selection,with suppression of replacement mutation in the FR. In contrast, the Rmutations in the CDR region of the same clones was no greater than maybe expected at random. Analysis of the matched light chain sequencesshowed that only clones 163.16 and 163.23 had significantly differentnumber of R mutations. In these instances the pattern mirrored the heavychain with negative selection occurring in the FR but no clear evidenceof antigen driven selection in the CUR1 and CDR2 regions.

Discussion

In this study, combinatorial antibody gene libraries and phage displayhave been used to isolate high affinity human Fab fragments withspecificity for the p53 tumour suppressor gene product. The isolatedFabs bound to the amino terminal region of p53 between residues 27 and44, and were reactive with both recombinant p53, and with mutant p53immunoprecipitated from colorectal cancer cells. This study representsthe first report of the isolation of anti-p53 antibodies from anindividual with a demonstrable antibody response to p53. As such, itprovides important information on the gene usage and epitope specificityof the immune response to p53 seen in humans with malignancy. It alsoprovides a reproducible strategy for the exploitation of these as usefulimmunotherapeutic agents.

Antibody phage display techniques are being increasingly used to examinethe nature and specificity of the humoral immune response to a range ofinfectious and autoimmune diseases. The occurrence of anti-p53antibodies in the serum of some individuals with colorectal cancerprovided an opportunity to more closely examine the specificity of thisresponse to an important tumour suppressor gene product.

In this study, libraries were constructed from pericolic lymph nodesdraining a colorectal tumour, since it was considered that this tissuewas more likely to represent an enriched source of anti-p53 antibodies.In order to further increase the likelihood of isolating specific Fabs,we selected individuals with a demonstrable IgG1 response to p53 proteinor a portion thereof. In this regard, it is of note that all those Fabswith high affinity for p53 were derived from the individual with thehighest serum antibody titre against p53, and that no antibodies wereisolated from the one individual without a demonstrable serum response.

This study has, for the first time, provided an opportunity to examinethe genetic structure of naturally occurring p53 antibodies, and to drawinferences from that structure regarding the nature of the immuneresponse that produced them.

Nucleotide sequencing showed that the V genes of the p53 Fabs hadundergone extensive mutation (14-18.5%), a finding that was highlyunlikely to be explained on the basis of Tth (polymerase) induced errors(12). In fact, this frequency of V gene mutations is higher than thatreported for class switched germinal centre and memory B cells (up to4%), and strongly supports that the isolated antibodies reflect theoccurrence of a specific antigen-driven humoral immune response in theseindividuals. The particularly high mutation frequency may reflect thechronic nature of antigen exposure in individuals with malignancy. Whilethe mechanism of p53 presentation to the immune system remainsuncertain, it is clear that the process can develop early in the processof tumor development. For instance, serum p53 antibodies have beenreported in smokers several years prior to the detection of themalignancy (13). This suggests that antigenic p53 may be presented tothe immune system throughout the course of the disease, and that thiscontinual exposure may be responsible for the extensive somatic mutationrate in the V genes.

Statistical analysis of the frequency of replacement mutations in the Vgenes provides further evidence to support the contention that theisolated Fabs arose as a result of antigen-driven selection Negativeselection for the replacement mutations was seen in the frameworkregions of VH1 family antibodies, and their positive selection in CDR1and 2 of clone 163.17, are typical features of affinity maturedantibodies.

The structural features of the Fabs, and the inferences drawn from them,are supported by affinity analysis using surface plasmon resonance. Theisolated Fabs all showed relatively high affinity for denatured p53,again suggesting that they represent the product of a specificantigen-driven immune response.

The successful isolation of stable and clonal Fabs has also allowed acloser examination of the epitope specificity of naturally occurring p53antibodies. Fabs isolated in this study bound to residues 27-44 of p53,a region which is predominantly specific to human p53 (14). This regionis particularly important as a site for interaction with transcriptionmachinery, as well as viral proteins (15). To date, most polyclonalserum antibodies and murine monoclonals against p53 have been shown tobind to a narrow range of immunodominant epitopes that span in residuesin the N-terminal region (10-25, 40-50), the central region (120-130,205-215, 285-295) and the C-terminal region (345-393). This studydemonstrates that lymphocytes from individuals with cancer represent aunique and valuable source of such antibodies, and outlines strategiesfor the successful exploitation of this important resource.

Example 2 Isolation and Characterisation of Anti-p53 Antibodies to theCentral Domain of p53 Materials & Methods Purification of p53 Centraland Carboxy Terminal Domain

The central carboxy terminal domains of whole p53 (central domain,residues 95-298; carboxy terminal domain, 283-393) was amplified fromwild type p53 sequence by PCR using p53 specific primers (centralforward, 5′-ggccccatatgtcttctgtcccttcccag; central reverse,5′-agtcatatgtcacagctcgtggtcaggctc; carboxy forward,5′-gagaccatatgacagaggaacagaatctc; carboxy reverse,5′-agtcatatgtcagtctgagtcaggccc). The PCR products were digested withNde1 and cloned into the Nde1 site of the bacterial expression vectorpET19b (Novagene). The central and carboxy terminal domains wereexpressed and purified as described for whole p53.

Purified central domain and carboxy terminal domain protein was used forthe identification of colorectal cancer patients with central or carboxyterminal domain specific serum antibodies, the biopanning of Fabantibody, and the detection of soluble central domain reactive Fabs. Theprotocols to do this are essentially the same as those described inExample 1, except the central domain or carboxy terminal domain wascoated onto the wells of microtitre plates at 2 μg/ml in PBS.

The antigens used in the cross reactivity ELISA were coated at the sameconcentrations as described above (Example 1) except for the carboxyterminal domain of the p53 which was coated at 2 μg/ml in carconatebuffer and early pregnancy factor (EPF) which was coated at 2 μg/ml inPBS.

Immunoblot Analysis

Purified whole p53 and central domain run on SDS/PAGE gels wereelectroblotted onto PVDF membranes (0.22 μM, NEN/Dupont) for one hour at100 V (Towbin et al, 1979) and blocked for one hour in 10% skim milkpowder/PBS The primary antibodies DO7 (0.25 μg/ml in 10% skim milkpowder/PBS), Pab240 (DO7 (0.25 μg/ml in 10% skim milk powder/PBS), 163.1Fab bacterial supernatant or 1159.8 bacterial supernatant were thenincubated for 1 hour at room temperature. Following this, the membranewas washed three times in PBS (10 minutes each wash), incubated for onehour with alkaline phosphatase goat anti-mouse or alkaline phosphatasegoat anti human, Fab specific (0.1 μg/ml in 10% skim milk powder/PBS)and then washed three time with PBS. The membrane was equilibrated withcarbonate buffer for five minutes prior to the addition of an alkalinephosphatase substrate solution (NBT [0.3 μg/ml], BCIP [0.15 μg/ml] incarbonate buffer pH 9.6).

Results Isolation of Central Domain Reactive Clones

Five rounds of biopanning were performed against pure p53 central domainprotein. The soluble Fab from clones taken from the final round ofpanning (n=88) were assessed for reactivity to the protein by ELISA Itwas found that a single clone (1159.8) was reactive to the centraldomain and to whole p53 (FIG. 11). Further analysis revealed that 1159.8did not react with a range of other antigens including tumour antigens,self proteins, foreign proteins and haptens and confirms this clonesspecificity to central domain of p53 (FIG. 12).

The ability of the clone to bind to whole p53 and the central domain wasalso assessed by immunoblot (FIG. 13). It was shown that 1159.8 bound towhole p53 and central domain in a similar manner to the murinemonoclonal antibodies DO7 and Pab 240. These murine monoclonalantibodies are specific for the amino terminal and central domains ofp53 respectively. No central domain reactivity was observed with theamino terminal reactive Fab clone, 163.1, confirming the specificity of1159.8 to this domain. The nucleotide sequence and the deduced aminoacid sequence of clone 1159.8 can be found in SEQ ID No 29 and 30 for1159.8 light chain variable sequence and 1159.8 heavy chain variablesequence respectively.

Example 3 Mammalian Expression Vector Construction of MCO1

Equal concentrations (1 μg) of two synthetic oligonucleotides, 99 mer(sense: CT AGT GGC CAG GCC GGC CAG GAA CAA AAA CTC ATC TCA GAA GAG GATCTG AAT CGG GCC GCA TAG TTC CCC GCG GCT GCT CAC TAT ACG CGC CAG GAG G)and 91 mer (antisense: CTC GCG CGT ATA GTG AGC ACC CCC GGG GAA CTA TGCGGC CCC ATT CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC CTG GCC GGC CTG GCCA) were annealed in Sequanase reaction buffer (USB) by heating at 75° C.for 2 minutes followed by cooling to 35° C. over 1.5 h. Thedouble-stranded oligonucleotide (30 pmole) was then phosphorylated byincubating in 10 mM ATP, 1× polynucleotide kinase buffer and 10 Upolynucleotide kinase (Boehringer Mannheim) at 27° C. for 60 min. Thekinase was inactivated and the DNA was phenol extracted, ethanolprecipitated and resuspended in 20 μl water. The double-strandedoligonucleotide was then ligated into phosphatase-treated, Spe1/BstX1digested NPC3, and the construct was transformed into electrocompetentXL1-Blue E. coli cells (Stratagene). Clones containing the syntheticoligonucleotide cassette were then identified by restriction enzymeanalysis (MCO1 contains a Sma1 site which is not present in NPC3), andby nucleotide sequencing (Sanger).

Example 4 Construction of Whole Antibody Materials & Methods

The strategy for cloning and production of whole antibody is shown inFIG. 5. The heavy and light chain variable regions from selected Fabclones were amplified by PCR using a touch down protocol. A typicalreaction included template DNA (100 ng), 50 pmol each PCR primer (heavychain primers, p53Vhfor-5′-ata gtt gcg gcc gct gtg cag ctg ctc gag andp53Vhrev 5′-agt ttc act agt tga gga gac ggc; light chain primers,p53Vkfor 5′-tta cat gtc gac gcg gcc gag ctc acc and p53Vkrev:5′-ccc tggttc gac ctt tag ttt aga tct act gat), 1.5 mM MgCl₂, 200 μM dNTP's. 0.12U Pfu polymerase in a volume of 50 μl. The conditions for the PCR were94° C. for 4 minutes and then 2 cycles of 94° C. (denaturing) for 30 s,65° C. for 1 minute (primer annealing), 72° C. for 90 seconds (primerextension). An additional 20 cycles were then carried out under the sameconditions except that the annealing step was reduced by 0.5° C. witheach successive round. A final 10 PCR cycles were then performed with anannealing temperature of 55° C.

The heavy and light PCR products were electrophoresed on 0.8% (w/v)Nusieve agarose, purified using Qiaquick DNA purification columns(Qiagen) and digested with appropriate restriction endonucleases (heavychain, Not 1 and Spe 1, light chain Sal 1 and Xba 1). The purified heavychain was then cloned into the heavy chaip expression vector pG1D105 andthe light chain into the light chain expression vector pKN101 (FIG. 5).The sequence of individual heavy and light chain clones was determinedby cycle sequencing according to the manufacturer's specifications(Promega). Selected heavy and light chain clones were grown in XL1-blueE. coli, the plasmid extracted by alkaline lysis and then doublepurified by CsCl gradient centrifugation.

Transfection and Selection of an Antibody Expressing CHO Cell Line

The parent cell line, CHO DG44, was seeded into 10 cm dishes at 6×10⁵cells/dish and grown in CHO-S-SFM II (Gibco) supplemented with HT (10 mMsodium hypoxanthine, 1.6 mM thymidine). The vectors containing the heavychain (pG1D102) and light chain (pKN100) were transfected into the CHODG44 cells using the Fugene transfection reagent (Boehringer Mannheim)according to the manufacturers specifications. In brief, equal amountsof heavy and light chain vector (1-5 μg of DNA total), were added toFugene at a DNA:Fugene ratio of 1:3, and allowed to mix for 10 minutesat room temperature. The Fugene/DNA mix was added dropwise to the mediaand cells, and then incubated overnight.

Transfected cells were passaged into CHO-S-SFM-II containingmethotrexate (5 nM) and G418 (500 μg/ml) and pools of transfectantswhich expressed antibody were subsequently plated at a cellconcentration of 0.5 cells/well. The concentration of methotrexate wasincreased with each passage (5-50 nM) and the level of G418 wasgradually removed from the culture media over the same period of time.Specific clones were expanded and then subjected to another round ofcloning at 0.5 cells/well to ensure that a clonal population of cellswas generated. The clones selected for the production of whole anti-p53antibody (A1, B4) were maintained in media without G418 or methotrexate.

Whole Antibody Production and Purification

Selected cell lines were cultured in cell culture and spinner flasks(1×10⁵-2×10⁶ cells/ml) over 5 to 10 days, the culture media and cellswere then centrifuged, firstly at 1200 g to remove most cells and thenagain for 15 minutes at 4000 g to remove other cellular debris.

Antibody was purified from the culture supernatant using Protein Aaffinity chromatography. In brief, a column containing 1 ml of Protein A(BioRad AffiPrep Protein A resin) was equilibrated with 50 ml of PBS andthe supernatant run over the column under gravity. The resin was thenwashed with 10 ml PBS under gravity. The antibody was eluted with 10 mlof 0.1 M glycine/0.2 M NaCl pH 3 and collected into 0.5 ml fractions,each of which was adjusted to pH 7 with 50 μl of 1M Tris HCl, pH 8.Purified antibody was used for all antibody characterisation.

Purified whole antibody was detected and quantified using an anti-humanantibody capture ELISA. Wells of microtitre plate were coated with 100μl of a goat anti-human IgG Fc specific antibody (1.8 μg/ml in 0.1MNaHCO₃/0.1% Bronidox) overnight at 4° C. The wells were then washed with200 μl of PBS/0.05% Tween/0.1% Bronidox three times, and blocked withPBS/1% skim milk/0.5% BSA/0.1% Bronidox for 1 hour at 37° C. Serialdilutions of human antibody (1:2 to 1:2000) were applied to the wellsand incubated for 1 hour 37° C. The wells were then washed three timeswith PBS/0.05% Tween/0.1% Bronidox and the wells incubated with 100 μlof a biotin conjugated anti-human Kappa (0.05 μg/ml in PBS/1% skimmilk/0.5% BSA/0.1% Bronidox; Rockland, Mass., USA) for 30 minutes at 37°C. Wells were washed three times and 100 μl of alkaline phosphataseconjugated streptavidin (0.04 mg/ml in PBS/1% skim milk/0.5% BSA/0.1%Bronidox; Jackson Immuno Research Lab Inc) added to each well for 30minutes at 37° C. After further washing, p-nitrophenyl alkalinephosphatase substrate (1 mg/ml in carbonate buffer; Sigma) was added,the colour allowed to develop for approximately 20 minutes and theabsorbance read at 410 nm. The concentration of antibody was calculatedby comparing the absorbance value of the unknown to a standard curvethat was generated using a positive control IgG1 antibody (The BindingSite, UK) diluted at concentrations ranging from 65-0.4 ng/ml.

Characterisation of Whole Antibody ELISA Analysis of Anti-p53 Antibodies

Purified p53 (100 μl at 2 μg/ml in PBS), the central domain of p53 (100μl at 2 μg/ml in PBS) and C-terminal domain (100 μl at 2 μg/ml incarbonate buffer) were coated onto wells of a microtitre plate overnightat 4° C. Wells were washed three times with 200 μl of PBS and then 100μl of purified antibody was applied in duplicate and incubated for 2hours at room temperature. After washing with PBS, 100 μl of HRPconjugated goat anti-human (0.5 μg/ml in PBS/2% BSA. Jackson ImmunoResearch Lab Inc) antibody was added. After further washing, colour wasdeveloped with 100 μl of TMB substrate (Kirkegaard and PerryLaboratories, Gaithersburg Md.) and the reaction was stopped with 50 μlof 1 M H₂SO₄. The monoclonal antibody DO7 (0.1 μg/ml) was used as apositive control under similar conditions except this antibody wasdetect with a HRP conjugated goat anti-mouse (0.5 μg/ml in PBS/2% BSA:Jackson Immunq Research Lab Inc).

The reactivity of p53 antibodies with other antigens was assessed byELISA using a similar method to that described above for p53. Thefollowing antigens and concentrations were used in coating of wells;insulin (5 μg/ml), erbB2 extracellular domain (2 μg/ml), CEA (1 μg/ml),BSA (20 μg/ml; Sigma-Aldrich, Castlehill, Australia), keyhole limpethaemocyanin (1 μg/ml; Sigma-Aldrich), BSM (10 μg/ml;Sigma-Aldrich.Castlehill, Australia), hen egg white lysozyme (10 μg/ml;Sigma-Aldrich,Castlehill, Australia) and early pregnancy factor (10 μg/ml).

Epitope Analysis Construction of p53 Gene-Fragment Libraries

The p53 gene was PCR amplified from the pET19b/p53 construct (7), run ona 0.8% Nusieve gel and purified using Oiaquick DNA purification columns.The gene fragment was digested with DNase I (0.5 U/ml) and p53 genefragments between 100-300 base pairs were gel-purified and end-repairedusing T₄ DNA polymerase. Phosphorylated linkers FUSP (16) were blunt-endligated to the p53 gene fragments and the ligated products were PCOamplified in a 75 μl mixture containing 12.5 μl 10×PCR buffer (FischerBiotech, Perth, Australia), 12.5 μl 25 mM MgCl₂, 20 μl 1.25 mM dNTP, 2.5U Tth polymerase (Fischer Biotech, Perth, Australia) and 250 pmol ofeach of the primers: MCO234 (5′-atatccagacgctggcggtg) and MCO235(5′-atatccagcagctggcggtg). The PCO reaction was performed using a PerkinElmer GeneAmp PCS System 9600 starting with 94° C. for 5 minutes,followed by 35 cycles of amplification at 94° C. for 1 minutes, 60° C.for 30 s, 72° C. for 30 s and then a final incubation at 72° C. for 10minutes. The PCR amplified fragments were gel purified, digested withPfl MI (5 u/μg of DNA) and were ligated to Sfi I (5 u/μg DNA) digestedfUSE5 vector DNA (17). The ligation mixture was electrotransformed intoMC1061 using Blo-Rad Gene Pulser. To determine the library size,aliquots of 1 and 10 μl of the culture were plated on Luria broth agarplate containing tetracycline (20 μg/ml) and streptomycin (50 μg/ml).The remaining culture was grown overnight in 400 ml 2YT supplementedwith tetracycline (20 μg/ml) and streptomycin (50 μg/ml). Phage werepurified and titred as described by Smith (18).

Affinity Selection of p53 Gene Fragment Libraries

Wells of a microtitre plate were coated overnight at 4° C. with 100 μlof anti-p53 antibody (10 μg/ml) in PBS. The solution was removed, thewell washed 3 times with 0.05% Tween/PBS, and then blocked with PBScontaining 0.05% Tween and 2% skim milk at 37° C. for 1 hour. p53 genefragment libraries diluted to 10¹¹ TU/100 μl in PBS containing 0.05%Tween and 2% skim milk were added and the plate was incubated at 37° C.for 1 hour. Unbound phage were removed by washing five times in 0.05%Tween/PBS, and five times in PBS/0.5% Tween. Bound phage were eluted for10 minutes with 100 μl of 0.1 M glycine, pH 3.0 at room temperature andthe eluate was immediately neutralised with 10 μl of 1M Tris-HCl, pH9.5. The neutralised phage solution was used to infect 1 ml of mid-logK91Kan culture for 15 minutes at 37° C. The culture was incubated foranother 30 minutes at 37° C. in 10 ml 2 YT containing tetracycline (0.2μg/ml). Aliquots of 100, 1 and 0.01 μl of culture were plated onto Luriabroth agar plate containing tetracycline (20 μpg/ml) and kanamycin (100μmg/ml) to determine the colony forming units of each aliquot and theafter infection titre. The remaining infected culture was added to 100ml of 2 YT containing tetracycline (20 μg/ml) and kanamycin (100 μg/ml)and then incubated overnight with shaking to amplify the library. Phagewere precipitated and were used for the second round of panning. Threerounds of panning were performed.

Screening of Positive Clones by Phage ELISA

Single colonies from the outputs of each round of panning were grownovernight and phage isolated Wells of a microtitre plate were coatedwith the anti-p53 antibody under the same conditions used for panning.Freshly prepared phage (10¹⁰-10¹¹ cfu/ml) were incubated for 1 hour oncoated and non-coated wells and then washed with 0.05% TweenlPBS Afterwashing with PBS, 100 μl HRP conjugated mouse anti-M13 (0.5 μg/ml inPBS/2% BSA: Pharmacia) antibody was added and incubated for 1 hour atroom temperature. After further washing, colour was developed with 100μl of TMB substrate (Kirkegaard and Perry Laboratories Gaithersburg Md.)and the reaction was stopped with 50 μl of 1 M H₂SO₄.

Immunoprecipitation

The following cell lines were used for immunoprecipitation: HT29 (humancolorectal cancer cell line with mutant p53), MCF-7 (breast cancer cellline with wild type p53) and MethA (mouse tumour cell line with mutantp53). Approximately 10⁷ cells were lysed in TNES buffer (50 mM Tris pH7.5, 2 mM EDTA, 100 mM NaCl, 1% Nonidet P40, protease inhibitor cocktail(Boehringer Mannheim) and 1 mM PMSF), and then cell debris was removedby centrifugation at 10000 g for 10 minutes. Approximately 250 μg oftotal protein from the cell lysate was used in each immunoprecipitation.Either the monoclonal antibody DO7 (0.5 μg) or the whole anti-p53antibody (approximately 1 μg/ml) was added to the lysate and incubatedfor one hour at 4° C. At this point 20 μl (packed volume) of proteinA-sepharose (Zymed Laboratories Inc.) was added to all tubes andincubated for a further one hour at 4° C. The protein A sepharose waswashed four times with PBS, and subject to 10% SDS PAGE under denaturingand reduced conditions. Proteins were transferred to PVDF membrane byelectroblotting, blocked with 10% skim milk powder and probed with agoat anti-human p53 antibody specific for the N-terminal region of theprotein (0.2 μg/ml). This was followed by four washes with PBS, one hourincubation with a HRP conjugated donkey anti-goat-antibody (0.2 μg/ml)and four more washes with PBS. The blots were developed usingchemiluminescence substrate (DuPont NEN) Protein A sepharose alone, andextract alone were each included in all experiments as negativecontrols.

Immunohistochemistry

4 μm paraffin sections of colorectal tumour tissue were dewaxed andrehydrated, treated with 3% (v/v) hydrogen peroxide for 5 minutes, andthen microwaved in 0.01 M citrate buffer pH 6.0 for 10 minutes. Thesections were then blocked with normal rabbit serum (1.5 in 2% BSA/TBS)for 20 minutes before incubating for one hour with either the wholeanti-p53 antibody (C4B4) or the monoclonal antibody DO7 (both diluted to2.5 μg/ml in 2% BSA/TBS). The bound antibody was then detected byincubating the slide for 30 minutes with horseradish peroxidaseconjugated rabbit anti human or horseradish peroxidase conjugated rabbitanti-mouse (1:100 in 2% BSA/TBS), each for 30 minutes. Each incubationwas followed by extensive washing in TBS. Colour was developed with3-3′diaminebenzidine tetra-hydrochloride (0.03% in 0.003% H₂O₂), andsections were counterstained with haematoxylin before mounting.

Results Construction and Characterisation of the Whole Antibody

The variable region genes cloned into the expression vectors pKN100 andpG1D102 were sequenced and shown to have the same sequence as the parentFab clones. CHO cells were successfully transfected with the vectors andtwo clones, C4B4 and C4A1 from the pools of transfectants. When analysedby ELISA, the antibodies from both clones were shown to react with wholep53 but not the central or C-terminal domains of the protein, indicatingthat they were reactive with the amino terminal region alone. Theantibody from clone C4B4 was purified using protein A chromatography andanalysed by SDS PAGE under reduced and non-reduced conditions and shownto produce a whole antibody with heavy and light chains of the correctsize (FIG. 6). Purified antibody was assessed by ELISA for crossreactivity with a range of antigens including haptens, self antigens andtumour antigens. It was found that the antibody C4B4 bound to whole p53but not to the other antigens used in the assay (FIG. 7). The antibodyproduction from C4B4 was higher than that obtained from C4A1, thereforethis clone was subjected to a final round of cloning to produce theclone referred to as C4B4G4.

Immunoprecipitation

The human cell lines HT29 and MCF-7, and the mouse cell line MethA wereused to assess the reactivity of the whole antibody to p53 derived fromeukaryotic cells. It was shown that C4B4 was able to immunoprecipitatewild and mutant human p53 and mutant murine p53 in the same manner asthe p53 monoclonal antibody DO7 (FIG. 8).

Epitope Analysis

The p53 gene fragment library was subjected to 3 rounds of pannningagainst purified C4B4 antibody. Analysis of phage clones from rounds twoand three for reactivity to the C4B4 showed that 15/22 clones in roundtwo and 19/22 from round three bound to the antibody. Sequence analysisof the reactive clones showed that they all contained the fragment ofp53 containing amino acid residues from 40-54 (FIG. 9). When compared tothe epitope defined in experiments using deletion mutants and the parentFab antibody 163.5, the epitope may be defined to include those residuesfrom 40-44.

Immunohistochemistry

Purified C4B4G4 antibody was used to detect p53 in 4 μm sections oftumour tissue and matched normal tissue from individuals with colorectalcancer. It was demonstrated that the antibody was reactive specificallywith tumour cells and that it had a nuclear staining pattern similar tothe positive control antibody DO7 (FIG. 10).

Example 5 p53 Detection Systems

The antibody or a fragment thereof of the present invention may be usedfor the detection of polypeptides encoded by the p53 gene invertebrates, in normal and in disease states. For example, the antibodymay be used to capture p53 protein from patient sample in the followingmanner.

The anti-p53 antibody or fragment thereof such as anti-p53 Fab, iscoated onto an appropriate surface (e.g., ELISA plate, Polysorb-immunoplate (NUNC, Denmark) using a solution of about 2 μg/ml. This is thenblocked with bovine serum albumin (BSA) at a concentration of about 2%(w/v) before the surface is washed with phosphate buffered saline (PBS).The patient sample is added and incubated for an appropriate length oftime before being removed and the surface again washed with PBS. A p53specific polyclonal antibody conjugated to a reporter molecule (e.g.,alkaline phosphatase, horse radish peroxidase, FITC) is then addedbefore the surface is again washed with PBS or other buffer A substrate(e.g., 5-bromo-4-chloro-3-indolyl phosphate (BCIP) with nitro bluetetrazolium (NBT), 3,3′,5,5′-tetramethylbenzidine dichloride (TMB))appropriate for the reporter molecule is then added in order tovisualise and, if necessary, quantitate bound p53 protein.

Example 6 Pharmaceutical Formulations

While it is of course possible for an antibody or fragment thereof ofthe present invention to be administered alone, it is preferable that itbe administered as a pharmaceutical formulation. The active ingredientmay comprise, for topical administration from 0.001% to 10% by weight,eg., from 1% to 5% by weight of the formulation, although it maycomprise as much as 10% by weight but preferably not in excess of 5% byweight, and more preferably from 0.1% to 1% by weight of theformulation.

The topical formulations of the present invention, comprise an activeingredient together with one or more acceptable carriers and optionallyany other therapeutic ingredients. The carriers must be “acceptable” interms of being compatible with the other ingredients of the formulation,and not deleterious to the recipient thereof.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions. These may be prepared by dissolving theactive ingredient in a aqueous solution of a bactericidal and/orfungicidal agent and/or any other suitable preservative, and optionallyincluding a surface active agent. The resulting solution may then beclarified by filtration, transferred to a suitable container andsterilised. Sterilisation may be achieved by: autoclaving or maintainingat 90° C.-100° C. for half an hour, or by filtration, followed bytransfer to a container by an aseptic technique Examples of bactericidaland fungicidal agents suitable for inclusion in the drops arephenylmercuric nitrate or acetate (0.002%), benzalkonium chloride(0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for thepreparation of an oily solution include glycerol, diluted alcohol andpropylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those described above in relation to thepreparation of drops. Lotions or liniments for application to the skinmay also include an agent to hasten drying and to cool the skin, such asan alcohol or acetone, and/or a moisturiser such as glycerol, or oilsuch as castor oil or arachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with a greasy or non-greasy basis. Thebasis may comprise hydrocarbons such as hard, soft or liquid paraffin,glycerol, beeswax, a metallic soap: a mucilage an oil of natural originsuch as almond, corn, arachis, castor or olive oil, wool fat or itsderivatives, or a fatty acid such as stearic or oleic acid together withan alcohol such as propylene glycol or macrogols.

The formulation may incorporate any suitable surface active agent suchas an anionic cationic or non-ionic surface active such as sorbitanesters or polyoxyethylene derivatives thereof. Suspending agents such asnatural gums, cellulose derivatives or inorganic materials such assilicaceous silicas, and other ingredients such as lanolin may also beincluded.

Further, it will be apparent to one of ordinary skill in the art thatthe optimal quantity and spacing of individual dosages of an antibody(or fragment thereof) of the present invention will be determined by thenature and extent of the condition being treated the form, route andsite of administration, and the nature of the particular vertebratebeing treated. Also, such optimum conditions can be determined byconventional techniques.

It will also be apparent to one of ordinary skill in the art that theoptimal course of treatment, such as, the number of doses of theantibodies (or fragments thereof) of the present invention given per dayfor a defined number of days, can be ascertained by those skilled in theart using conventional course of treatment determination tests.

The following are to be construed as merely illustrative examples offormulations and not as a limitation of the scope of the presentinvention in any way.

Example 6(a) Capsule Composition

A pharmaceutical composition containing the antibody(s) (or fragmentsthereof) of the present invention in the form of a capsule is preparedby filling a standard two-piece hard gelatin capsule with 50 mg of anantibody (or fragment thereof) of the invention, in powdered form, 100mg of lactose, 35 mg of talc and 10 mg of magnesium stearate.

Example 6(b) Injectable Parenteral Composition

A pharmaceutical composition of this invention in a form suitable foradministration by injection may be prepared by stirring 2% by weight ofan antibody (or fragment thereof) of the present invention in 10% byvolume propylene glycol and water. The solution is sterilised byfiltration.

Example 6(c) Ointment Composition

A typical composition for delivery as an ointment includes 1.0 g of theantibody (or fragment thereof) of the invention, together with whitesoft paraffin to 100.0 g is dispersed to produce a smooth, homogeneousproduct. Collapsible metal tubes are then filled with the dispersion.

Example 6(d) Topical Cream Composition

A typical composition for delivery as a topical cream is outlined below.

-   -   Antibody (or fragment thereof) 1.0 g    -   Polawax GP 200 25.0 g    -   Lanolin Anhydrous 3.0 g    -   White Beeswax 4.5 g    -   Methyl hydroxybenzoate 0.1 g    -   Deionised & sterilised Water to 100.0 g

The polawax, beeswax and lanolin are heated together at 60° C., asolution of methyl hydroxybenzoate is added and homogenisation isachieved using high speed stirring. The temperature is then allowed tofall to 50° C. The antibody (or fragment thereof) of the presentinvention is then added and dispersed throughout, and the composition isallowed to cool with slow speed stirring.

Example 6(e) Topical Lotion Composition

A typical composition for delivery as a topical lotion is outlinedbelow:

-   -   Antibody (or fragment thereof) 1.2 g    -   Sorbitan Monolaurate 0.8 g    -   Polysorbate 20 0.7 g    -   Cetostearyl Alcohol 1.5 g    -   Glycerin 7.0 g    -   Methyl Hydroxybenzoate 0.4 g    -   Sterilised Water about to 100.00 ml

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml of thewater at 75° C. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75° C. and added to the aqueous solution.The resulting emulsion is homogenised, allowed to cool with continuousstirring and the antibody (or fragment thereof) of the present inventionis added as a suspension in the remaining water. The whole suspension isstirred until homogenised.

Example 6(f) Eye Drop Composition

A typical composition for delivery as an eye drop is outlined below.

-   -   Antibody (or fragment thereof) 0.3 g    -   Methyl Hydroxybenzoate 0.005 g    -   Propyl Hydroxybenzoate 0.06 g    -   Purified Water about to 100.00 ml

The methyl and propyl hydroxybenzoates are dissolved in 70 ml purifiedwater at 75° C., and the resulting solution is allowed to cool. Theantibody (or fragment thereof) of the invention is then added, and thesolution sterilised by filtration through a membrane filter (0.022 μmpore size), and aseptically packed into sterile containers.

Example 6(g) Composition for Inhalation Administration (I)

For an aerosol container with a capacity of 20-30 ml, a mixture of 10 mgof an antibody (or fragment thereof) of the present invention with0.5-0.8% by weight of a lubricating agent, such as polysorbate 85 oroleic acid, and mixture was dispersed in a propellant, such as freon,and put into an appropriate aerosol container for either intranasal ororal inhalation administration.

Example 6(h) Composition for Inhalation Administration (II)

For an aerosol container with a capacity of 20-30 ml, a mixture of 10 mgof an antibody (or fragment thereof) of the present invention in ethanol(8-10 ml) 0.1-0.2% of a lubricating agent, such as polysorbate 85 oroleic acid was added, and the mixture dispersed in a propellant, such asfreon, and put into an appropriate aerosol container for eitherintranasal or oral inhalation administration.

Example 6(i) Composition for Parenteral Administration

The antibodies (or fragments thereof) and pharmaceutical compositions ofthe present invention are also useful for parenteral administration,that is, subcutaneously, intramuscularly or intravenously.

The compositions for parenteral administration will commonly comprise asolution of an antibody (or fragment thereof) of the present inventionor a cocktail thereof dissolved in an acceptable carrier such as water,buffered water, 0.4% saline and 0.3% glycine etc, wherein such solutionsare sterile and relatively free of particulate matter. These solutionsare then subsequently sterilised.

The compositions may contain further pharmaceutically acceptablesubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, etc. The concentration of theantibody (or fragment thereof) of the present invention in such acomposition can vary, and will be primarily based on fluid volumes,viscosities etc., according to the particular mode of administrationselected.

Thus, a pharmaceutical composition of the present invention forintramuscular injection could be prepared to contain 1 mL sterilebuffered water, and 50 mg of an antibody (or fragment thereof) of thepresent invention.

Similarly, a pharmaceutical composition for intravenous infusion maycomprise 250 ml of sterile Ringer's solution, and 150 mg of an antibody(or fragment thereof) of the present invention. Methods for preparingparenterally administrable compositions are apparent to those skilled inthe art, and are described in more detail in, for example Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton Pa.,hereby incorporated by reference herein.

Also the antibodies (or fragments thereof) of the present invention canbe lyophilised for storage and reconstituted prior to use.

Depending on the intended result the pharmaceutical composition of thepresent invention can be administered for prophylactic and/ortherapeutic treatments. In a therapeutic application compositions areadministered to a patient already suffering from a disease, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. In prophylactic applications, compositions containing theantibodies (or fragments thereof) or a cocktail thereof are administeredto a patient not already in a disease state to enhance the patient'sresistance.

Single or multiple administrations of the pharmaceutical compositionscan be carried out with dose levels and pattern being selected by thetreating physician. Regardless, the pharmaceutical composition of thepresent invention should provide a quantity of the altered antibodies(or fragments thereof) sufficient to effectively treat the patient.

It should also be noted that the antibodies of this invention may beused for the design and synthesis of either peptide or non-peptidecompounds (mimetics) which would be useful in the same therapy as theantibody (or fragment thereof).

TABLE I Clinical details, anti-p53 serum titre, and antibody librarysizes of patients selected for the study Detection of over- Anti-Predominant Patient Site of the Dukes Degree of expressed P53 P53 titreIgG Library ID Sex tumour Stage differentiation p53 mutation (IgG)isotype size 100 M sigmoid B Poor yes 0 NA 1.3 * 10⁶ colon 107 F sigmoidB Poor yes 512 IgG1 1.7 * 10⁶ colon 149 M rectum C Moderate yes 1024IgG1 1.6 * 10⁷ 163 M sigmoid B Poor yes 8192 IgG1 4.5 * 10⁷ colon 357 Frectum C Moderate no 512 IgG1 2.4 * 10⁷ 790 M sigmoid C Moderate yes16384 IgG1 3.0 * 10⁷ colon

TABLE II The most homologous germline sequence is shown together withthe number of nucleotide mutations. Nucleotide Nucleotide Clone VH Dmutations in VK gene mutations in number gene gene J gene the V region*family J gene the V region* 163.16 DP-7 ND JH4b 43/294 (14.6) DPK24 JK210/305 (3.2) 163.23 DP-7 ND JH4b 43/294 (14.6) DPK24 JK2 10/305 (3.2)163.22 DP-7 ND JH4b 44/294 (15) DPK24 JK2 11/305 (3.6) 163.1 DP-7 NDJH4b 44/294 (15) DPK24 JK2 11/305 (3.6) 163.15 DP-7 ND JH4b 45/294(15.3) DPK24 JK4  3/305 (1) 163.20 DP-7 ND JH4b 52/294 (17.7) DPK24 JK214/305 (4.6) 163.5 DP-7 ND JH4b 52/294 (17.7) DPK24 JK2 18/305 (5.9)163.7 DP-7 ND JH4b 51/294 (17.3) DPK24 JK4  7/305 (2.3) 163.6 DP-7 NDJH4b 51/294 (17.3) DPK24 JK4  6/305 (2) 163.9 DP-7 ND JH4b 50/294 (17)DPK24 JK2 14/305 (4.6) 163.2 DP-7 ND JH4b 52/294 (17.7) DPK24 JK4  4/305(1.3) 163.14 DP-7 ND JH4b 52/294 (17.7) DPK24 JK2 14/305 (4.6) 163.24DP-7 ND JH4b 54/294 (18.5) DPK24 JK2  0/305 (0) 163.17 DP-7 ND JH4b54/294 (18.5) DPK24 JK4  2/294 (0.6) *The number of nucleotide mutationsin the V region/total number of nucleotides (%)

TABLE IIIA Variable gene mutational analysis: The total number ofreplacement (R) and silent (S) mutations in the FR and CDR regions 1 and2 of each heavy chain genes. Total FR R CDR R CDR Clone number of Rmutations mutations FR R:S number mutations (expected) (expected) R:Sratio ratio p (FR)* p (CDR)* 163.16 35 15 (23.12) 8 (7.13) 15:8 8:2 0.020.13 163.23 35 15 (21.34) 8 (6.40) 15:8 8:2 0.02 0.13 163.22 36 15(21.34) 8 (6.58) 15:8 8:3 0.01 0.13 163.1 36 15 (21.34) 8 (6.58) 15:88:3 0.01 0.13 163.15 37 15 (21.94) 8 (6.76) 15:9 8:3 0.01 0.14 163.20 3816 (21.34) 7 (6.95) 16:9 7:6 0.01 0.16 163.5 38 16 (22.53) 7 (6.9) 16:97:6 0.01 0.16 163.17 39 16 (23.12) 7 (7.13) 16:10 7:6 0.01 0.16 163.6 3616 (21.34) 7 (6.58) 16:9 7:6 0.03 0.16 163.9 37 16 (21.94) 7 (6.77) 16:87:6 0.02 0.16 163.2 36 16 (22.53) 7 (6.58) 16:9 7:6 0.03 0.16 163.14 3616 (21.34) 7 (6.58) 16:9 7:6 0.03 0.16 163.24 42 19 (24.90) 8 (7.68)19:8 8:5 0.02 0.15 163.17 39 16 (23.14) 5 (7.13) 16:9 7:6 0.01 0.16*Shaded areas indicate clones with a non-random distribution of Rmutations.

TABLE IIIB Variable gene mutational analysis: The total number ofreplacement (R) and silent (S) mutations in the FR and CDR regions 1 and2 of each light chain genes. Total number of FR R CDR R FR CDR R and Smutations mutations R:S R:S Clone number mutations (expected) (expected)ratio ratio p (FR)* p (CDR)* 163.16 6 0 (1.97) 4 (1.05) 0:2 4:0 0.040.09 163.23 6 0 (2.55) 4 (2.05) 0:2 4:0 0.04 0.09 163.22 7 1 (2.97) 5(2.38) 2:0 5:1 0.07 0.07 163.1 7 1 (2.98) 5 (2.38) 1:1 5:1 0.07 0.07163.15 2 0 (0.85) 1 (0.68) 0:0 1:1 0.33 0.44 163.20 10 3 (4.25) 7 (3.41)3:3 2:2 0.19 0.19 163.5 13 6 (5.53) 2 (4.43) 6:3 2:2 0.21 0.09 163.17 53 (1.70) 1 (1.36) 1:0 2:2 0.32 0.30 163.6 4 1 (1.70) 1 (1.36) 1:0 1:20.32 0.30 163.9 11 5 (4.68) 2 (3.75) 5:4 2:0 0.23 0.15 163.2 2 0 (0.85)1 (0.68) 0:0 1:1 0.33 0.45 163.14 11 5 (4.68) 2 (3.75) 5:4 2:0 0.23 0.15163.24 0 0 (0) 0 (0) 0:0 0:0 1 1 163.17 2 0 (0.85) 1 (0.68) 1:1 0:0 0.330.44 *Shaded areas indicate clones with a non-random distribution of Rmutations.

TABLE IVA Primers Primer Sequence Annealing site VH1a 5′-CAG GTG CAG CTCGAG CAG TCT GGG-3′ 5′ V FR1 of VH1 family VH3a 5′-GAG GTG CAG CTC GAGGAG TCT GGG-3′ 5′ V FR1 of VH3 family VH1f 5′-CAG GTG CAG CTG CTC GAGTCT GGG-3′ 5′ V FR1 of VH1 family VH2f 5′-CAG GTG CAG CTA CTC GAG TCGGG-3′ 5′ V FR1 of VH2 family VH3f 5′-GAG GTG CAG CTG CTC GAG TCT GGG-3′5′ V FR1 of VH3 family VH4f 5′-CAG GTG CAG CTG CTC GAG TCG GG-3′ 5′ VFR1 of VH4 family VH5f 5′-GAG GTG CAG CTC GAG CAG TCT GGA-3′ 5′ V FR1 ofVH5 family VH6f 5′-CAG GTA CAG CTG CTC GAG TCA GGT CCA-3′ 5′ V FR1 ofVH6 family CG1Z 5′-GCA TGT ACT AGT TTT GTC ACA AGA TTT GGG-3′ 3′ primer,γ1 hinge region (reverse transcription primer) CG2Z 5′-CGG TGG ACT AGTGAC ACA ACA TTT GCG 3 primer, γ2 hinge region (reverse transcriptionprimer) CG3Z 5′-TGG GCA ACT AGT GCA TGT GTG AGT TGT G 3′ primer, γ3hinge region (reverse transcription primer) CG4Z 5′-TGG GCA ACT AGT GCATGG GGG ACC ATA TTT GGA 3 primer, γ4 hinge region (reverse transcriptionprimer) Primers used for the reverse transcription and amplification ofheavy chain immunoglobulin genes. Nucleic acid residues that are in boldrepresent restriction enzyme sites

TABLE IVB Primers Primer Sequence Annealing site VK1a 5′-GAC ATC GAG CTCACC CAG TCT CCA-3′ 5′ V FR1 of Vκ1 family VK2a 5′-GAT ATT GAG CTC ACTCAG TCT CCA-3′ 5′ V FR1 of Vκ2 family VK3a 5′-GAA ATT GAG CTC ACG CAGTCT CCA-3′ 5′ V FR1 of Vκ3 family CK1Z 5′-GCG CCG TCT AGA ATT AAC ACTCTC CCC 3′ primer, 3′ end of κ light TGT TGA AGC TCT TTG TGA CGG GCG AACchain (reverse transcription TCA G-3′ primer) CL2 5′-cgc cgt cta gaa ctatga aca ttc tgt 3′ primer, 3′ CL region of agg human lambda light chain(reverse transcription primer) VH-2 5′-CAG TCT GAG CTC ACT CAG CCR CCCFR1 of human lambda light chain VL1 and VL2 families VL3 5′-TCC TAT GAGCTC ACT CAG FR1 of human lambda light chain VL3 family VL4-5-9 5′-CAGCCT GAG CTC ACT CAG FR1 of human lambda light chain VL4 VL5 and VL9families VL6 5′-AAT TTT GAG CTC ACT CAG CCC FR1 of human lambda lightchain VL6 family VL7 5′-CAG GCT GAG CTC ACT CAG GAG FR1 of human lambdalight chain VL7 family VL8 5′-CAG ACT GAG CTC ACC CAG GAG FR1 of humanlambda light chain VL8 family VL10 5′-CAG GCA GAG CTC ACT CAG CCA FR1 ofhuman lambda light chain VL10 family Primers used for the reversetranscription and amplification of the human k light chainimmunoglobulin genes. Nucleic acid residues in bold representrestriction enzyme sites

TABLE V Identification of individual sequences Light or SEQ ID No CloneType Heavy Chain 1 163.1 DNA Light 2 163.1 DNA Heavy 3 163.2 DNA Light 4163.2 DNA Heavy 5 163.5 DNA Light 6 163.5 DNA Heavy 7 163.6 DNA Light 8163.6 DNA Heavy 9 163.7 DNA Light 10 163.7 DNA Heavy 11 163.9 DNA Light12 163.9 DNA Heavy 13 163.14 DNA Light 14 163.14 DNA Heavy 15 163.15 DNALight 16 163.15 DNA Heavy 17 163.16 DNA Light 18 163.16 DNA Heavy 19163.17 DNA Light 20 163.17 DNA Heavy 21 163.20 DNA Light 22 163.20 DNAHeavy 23 163.22 DNA Light 24 163.22 DNA Heavy 25 163.23 DNA Light 26163.23 DNA Heavy 27 163.24 DNA Light 28 163.24 DNA Heavy 29 1159.8 DNALight 30 1159.8 DNA Heavy 31 163.1 Amino acid Light 32 163.1 Amino acidHeavy 33 163.2 Amino acid Light 34 163.2 Amino acid Heavy 35 163.5 Aminoacid Light 36 163.5 Amino acid Heavy 37 163.6 Amino acid Light 38 163.6Amino acid Heavy 39 163.7 Amino acid Light 40 163.7 Amino acid Heavy 41163.9 Amino acid Light 42 163.9 Amino acid Heavy 43 163.14 Amino acidLight 44 163.14 Amino acid Heavy 45 163.15 Amino acid Light 46 163.15Amino acid Heavy 47 163.16 Amino acid Light 48 163.16 Amino acid Heavy49 163.17 Amino acid Light 50 163.17 Amino acid Heavy 51 163.20 Aminoacid Light 52 163.20 Amino acid Heavy 53 163.22 Amino acid Light 54163.22 Amino acid Heavy 55 163.23 Amino acid Light 56 163.23 Amino acidHeavy 57 163.24 Amino acid Light 58 163.24 Amino acid Heavy 59 1159.8Amino acid Light 60 1159.8 Amino acid Heavy

REFERENCES

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1.-131. (canceled)
 132. An isolated and purified peptide, the peptidecomprising at least one complementarity determining region (CDR)sequence of a human antibody having binding affinity to a p53 protein orportion thereof, wherein the antibody includes a sequence selected fromthe group consisting of SEQ ID Nos. 31-60.
 133. The peptide according toclaim 132, wherein the peptide comprises a complementarity determiningregion sequence selected from the group consisting of: SNQSVLYNSNSKNYLA,QQYFSSPYT, GQYIH, VINPSGGSANYAPSFQG, LSQALKY, SSQSVLYSSNNKNYLA,QQYFSTPLT, IINPSGGSANYAPKFKG, LLQALKH, SSQSVLYSLNNKNYLA, QQYYTTPYT,QQYFSTRLT, WASTRQS, QQYYGTPYT, IINPSGGSAGYAPKFKG, QQYYRTPLT, QQYFSTPYT,QQYYSTPYT, IINPSGGSANYAPRFKG, LLQSLKH, SSQSLLHRNGYNYLD, LGSTRAS,MQGLQTPYT, DSAMT, FIRSKAYGAATAYAASMKG, and VKAGGPDY.


134. The peptide according to claim 132, wherein the peptide induces animmune response when administered to human.
 135. An antibody or antibodyfragment having a binding affinity to a p53 protein or portion thereof,wherein the antibody or fragment thereof comprises the peptide accordingto claim
 132. 136. The antibody or antibody fragment according to claim135, wherein the antibody or antibody fragment induced an idiotypicresponse.
 137. The antibody fragment according to claim 135, wherein thefragment is selected from the group consisting of: F_(V), F_(ab),F(ab)₂, scF_(V) (sincle chain F_(V)), dAb (single domain antibody),bi-specific antibodies, diabodies and triabodies.
 138. The antibodyfragment according to claim 137, wherein the fragment is animmunologically active fragment.
 139. An antibody directed against thepeptide according to claim
 132. 140. An isolated and purified nucleicacid sequence comprising a polynucleotide sequence encoding the peptideaccording to claim
 132. 141. The isolated and purified nucleic acidsequence according to claim 140, wherein the nucleic acid sequencecomprises a polynucleotide sequence encoding an antibody or antibodyfragment.
 142. A method for inducing an immune response against adisease characterized by expression of p53, comprising administering toa human an immunologically effective amount of the peptide according toclaim 132, or the antibody or antibody fragment according to claim 136.143. A method of treatment and/or prophylaxis of disease characterizedby expression of p53, wherein said method comprises administering to ahuman a therapeutically effective amount of the peptide according toclaim 132, or the antibody or antibody fragment according to claim 136.144. A pharmaceutical composition comprising the peptide according toclaim 132 or the antibody or antibody fragment according to claim 136,together with a pharmaceutically acceptable carrier, adjuvant and/ordiluent.